Novel Substitution Mutant Receptors and Their Use in a Nuclear Receptor-Based Inducible Gene Expression System

ABSTRACT

This invention relates to the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression. More specifically, this invention relates to novel substitution mutant receptors and their use in a Group H nuclear receptor-based inducible gene expression system and methods of modulating the expression of a gene in a host cell for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic organisms.

FIELD OF THE INVENTION

This invention relates to the field of biotechnology or geneticengineering. Specifically, this invention relates to the field of geneexpression. More specifically, this invention relates to novel nuclearreceptors comprising a substitution mutation and their use in a nuclearreceptor-based inducible gene expression system and methods ofmodulating the expression of a gene within a host cell using thisinducible gene expression system.

BACKGROUND OF THE INVENTION

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties. However, the citation ofany reference herein should not be construed as an admission that suchreference is available as “Prior Art” to the instant application.

In the field of genetic engineering, precise control of gene expressionis a valuable tool for studying, manipulating, and controllingdevelopment and other physiological processes. Gene expression is acomplex biological process involving a number of specificprotein-protein interactions. In order for gene expression to betriggered, such that it produces the RNA necessary as the first step inprotein synthesis, a transcriptional activator must be brought intoproximity of a promoter that controls gene transcription. Typically, thetranscriptional activator itself is associated with a protein that hasat least one DNA binding domain that binds to DNA binding sites presentin the promoter regions of genes. Thus, for gene expression to occur, aprotein comprising a DNA binding domain and a transactivation domainlocated at an appropriate distance from the DNA binding domain must bebrought into the correct position in the promoter region of the gene.

The traditional transgenic approach utilizes a cell-type specificpromoter to drive the expression of a designed transgene. A DNAconstruct containing the transgene is first incorporated into a hostgenome. When triggered by a transcriptional activator, expression of thetransgene occurs in a given cell type.

Another means to regulate expression of foreign genes in cells isthrough inducible promoters. Examples of the use of such induciblepromoters include the PR1-a promoter, prokaryotic repressor-operatorsystems, immunosuppressive-immunophilin systems, and higher eukaryotictranscription activation systems such as steroid hormone receptorsystems and are described below.

The PR1-a promoter from tobacco is induced during the systemic acquiredresistance response following pathogen attack. The use of PR1-a may belimited because it often responds to endogenous materials and externalfactors such as pathogens, UV-B radiation, and pollutants. Generegulation systems based on promoters induced by heat shock, interferonand heavy metals have been described (Wurn et al., 1986, Proc. Natl.Acad. Sci. USA 83: 5414-5418; Arnheiter et al., 1990, Cell 62:51-61;Filmus et al., 1992, Nucleic Acids Research 20: 27550-27560). However,these systems have limitations due to their effect on expression ofnon-target genes. These systems are also leaky.

Prokaryotic repressor-operator systems utilize bacterial repressorproteins and the unique operator DNA sequences to which they bind. Boththe tetracycline (“Tet”) and lactose (“Lac”) repressor-operator systemsfrom the bacterium Escherichia coli have been used in plants and animalsto control gene expression. In the Tet system, tetracycline binds to theTetR repressor protein, resulting in a conformational change thatreleases the repressor protein from the operator which as a resultallows transcription to occur. In the Lac system, a lac operon isactivated in response to the presence of lactose, or synthetic analogssuch as isopropyl-b-D-thiogalactoside. Unfortunately, the use of suchsystems is restricted by unstable chemistry of the ligands, i.e.tetracycline and lactose, their toxicity, their natural presence, or therelatively high levels required for induction or repression. For similarreasons, utility of such systems in animals is limited.

Immunosuppressive molecules such as FK506, rapamycin and cyclosporine Acan bind to immunophilins FKBP12, cyclophilin, etc. Using thisinformation, a general strategy has been devised to bring together anytwo proteins simply by placing FK506 on each of the two proteins or byplacing FK506 on one and cyclosporine A on another one. A synthetichomodimer of FK506 (FK1012) or a compound resulted from fusion ofFK506-cyclosporine (FKCsA) can then be used to induce dimerization ofthese molecules (Spencer et al., 1993, Science 262: 1019-24; Belshaw etal., 1996 Proc Natl Acad Sci USA 93: 4604-7). Gal4 DNA binding domainfused to FKBP12 and VP16 activator domain fused to cyclophilin, andFKCsA compound were used to show heterodimerization and activation of areporter gene under the control of a promoter containing Gal4 bindingsites. Unfortunately, this system includes immunosuppressants that canhave unwanted side effects and therefore, limits its use for variousmammalian gene switch applications.

Higher eukaryotic transcription activation systems such as steroidhormone receptor systems have also been employed. Steroid hormonereceptors are members of the nuclear receptor superfamily and are foundin vertebrate and invertebrate cells. Unfortunately, use of steroidalcompounds that activate the receptors for the regulation of geneexpression, particularly in plants and mammals, is limited due to theirinvolvement in many other natural biological pathways in such organisms.In order to overcome such difficulties, an alternative system has beendeveloped using insect ecdysone receptors (EcR).

Growth, molting, and development in insects are regulated by theecdysone steroid hormone (molting hormone) and the juvenile hormones(Dhadialla, et al., 1998, Annu. Rev. Entomol. 43: 545-569). Themolecular target for ecdysone in insects consists of at least ecdysonereceptor (EcR) and ultraspiracle protein (USP). EcR is a member of thenuclear steroid receptor super family that is characterized by signatureDNA and ligand binding domains, and an activation domain (Koelle et al.1991, Cell, 67:59-77). EcR receptors are responsive to a number ofsteroidal compounds such as ponasterone A and muristerone A. Recently,non-steroidal compounds with ecdysteroid agonist activity have beendescribed, including the commercially available insecticidestebufenozide and methoxyfenozide that are marketed world wide by Rohmand Haas Company (see International Patent Application No.PCT/EP96/00686 and U.S. Pat. No. 5,530,028). Both analogs haveexceptional safety profiles to other organisms.

The insect ecdysone receptor (EcR) heterodimerizes with Ultraspiracle(USP), the insect homologue of the mammalian RXR, and binds ecdysteroidsand ecdysone receptor response elements and activate transcription ofecdysone responsive genes (Riddiford et al., 2000). The EcR/USP/ligandcomplexes play important roles during insect development andreproduction. The EcR is a member of the steroid hormone receptorsuperfamily and has five modular domains, A/B (transactivation), C (DNAbinding, heterodimerization)), D (Hinge, heterodimerization), E (ligandbinding, heterodimerization and transactivation and F (transactivation)domains. Some of these domains such as A/B, C and E retain theirfunction when they are fused to other proteins.

Tightly regulated inducible gene expression systems or “gene switches”are useful for various applications such as gene therapy, large scaleproduction of proteins in cells, cell based high throughput screeningassays, functional genomics and regulation of traits in transgenicplants and animals.

The first version of EcR-based gene switch used Drosophila melanogasterEcR (DmEcR) and Mus musculus RXR (MmRXR) and showed that these receptorsin the presence of steroid, ponasteroneA, transactivate reporter genesin mammalian cell lines and transgenic mice (Christopherson et al.,1992; No et al., 1996). Later, Suhr et al., 1998 showed thatnon-steroidal ecdysone agonist, tebufenozide, induced high level oftransactivation of reporter genes in mammalian cells through Bombyx moriEcR (BmEcR) in the absence of exogenous heterodimer partner.

International Patent Applications No. PCT/US97/05330 (WO 97/38117) andPCT/US99/08381 (WO99/58155) disclose methods for modulating theexpression of an exogenous gene in which a DNA construct comprising theexogenous gene and an ecdysone response element is activated by a secondDNA construct comprising an ecdysone receptor that, in the presence of aligand therefor, and optionally in the presence of a receptor capable ofacting as a silent partner, binds to the ecdysone response element toinduce gene expression. The ecdysone receptor of choice was isolatedfrom Drosophila melanogaster. Typically, such systems require thepresence of the silent partner, preferably retinoid X receptor (RXR), inorder to provide optimum activation. In mammalian cells, insect ecdysonereceptor (EcR) heterodimerizes with retinoid X receptor (RXR) andregulates expression of target genes in a ligand dependent manner.International Patent Application No. PCT/US98/14215 (WO 99/02683)discloses that the ecdysone receptor isolated from the silk moth Bombyxmori is functional in mammalian systems without the need for anexogenous dimer partner.

U.S. Pat. No. 6,265,173 B1 discloses that various members of thesteroid/thyroid superfamily of receptors can combine with Drosophilamelanogaster ultraspiracle receptor (USP) or fragments thereofcomprising at least the dimerization domain of USP for use in a geneexpression system. U.S. Pat. No. 5,880,333 discloses a Drosophilamelanogaster EcR and ultraspiracle (USP) heterodimer system used inplants in which the transactivation domain and the DNA binding domainare positioned on two different hybrid proteins. Unfortunately, theseUSP-based systems are constitutive in animal cells and therefore, arenot effective for regulating reporter gene expression.

In each of these cases, the transactivation domain and the DNA bindingdomain (either as native EcR as in International Patent Application No.PCT/US98/14215 or as modified EcR as in International Patent ApplicationNo. PCT/US97/05330) were incorporated into a single molecule and theother heterodimeric partners, either USP or RXR, were used in theirnative state.

Drawbacks of the above described EcR-based gene regulation systemsinclude a considerable background activity in the absence of ligands andnon-applicability of these systems for use in both plants and animals(see U.S. Pat. No. 5,880,333). Therefore, a need exists in the art forimproved EcR-based systems to precisely modulate the expression ofexogenous genes in both plants and animals. Such improved systems wouldbe useful for applications such as gene therapy, large-scale productionof proteins and antibodies, cell-based high throughput screening assays,functional genomics and regulation of traits in transgenic animals. Forcertain applications such as gene therapy, it may be desirable to havean inducible gene expression system that responds well to syntheticnon-steroid ligands and at the same is insensitive to the naturalsteroids. Thus, improved systems that are simple, compact, and dependenton ligands that are relatively inexpensive, readily available, and oflow toxicity to the host would prove useful for regulating biologicalsystems.

Recently, Applicants have shown that an ecdysone receptor-basedinducible gene expression system in which the transactivation and DNAbinding domains are separated from each other by placing them on twodifferent proteins results in greatly reduced background activity in theabsence of a ligand and significantly increased activity over backgroundin the presence of a ligand (pending application PCT/US01/09050,incorporated herein in its entirety by reference). This two-hybridsystem is a significantly improved inducible gene expression modulationsystem compared to the two systems disclosed in applicationsPCT/US97/05330 and PCT/US98/14215. The two-hybrid system exploits theability of a pair of interacting proteins to bring the transcriptionactivation domain into a more favorable position relative to the DNAbinding domain such that when the DNA binding domain binds to the DNAbinding site on the gene, the transactivation domain more effectivelyactivates the promoter (see, for example, U.S. Pat. No. 5,283,173).Briefly, the two-hybrid gene expression system comprises two geneexpression cassettes; the first encoding a DNA binding domain fused to anuclear receptor polypeptide, and the second encoding a transactivationdomain fused to a different nuclear receptor polypeptide. In thepresence of ligand, the interaction of the first polypeptide with thesecond polypeptide effectively tethers the DNA binding domain to thetransactivation domain. Since the DNA binding and transactivationdomains reside on two different molecules, the background activity inthe absence of ligand is greatly reduced.

A two-hybrid system also provides improved sensitivity to non-steroidalligands for example, diacylhydrazines, when compared to steroidalligands for example, ponasterone A (“PonA”) or muristerone A (“MurA”).That is, when compared to steroids, the non-steroidal ligands providehigher activity at a lower concentration. In addition, sincetransactivation based on EcR gene switches is often cell-line dependent,it is easier to tailor switching systems to obtain maximumtransactivation capability for each application. Furthermore, thetwo-hybrid system avoids some side effects due to overexpression of RXRthat often occur when unmodified RXR is used as a switching partner. Ina preferred two-hybrid system, native DNA binding and transactivationdomains of EcR or RXR are eliminated and as a result, these hybridmolecules have less chance of interacting with other steroid hormonereceptors present in the cell resulting in reduced side effects.

The EcR is a member of the nuclear receptor superfamily and classifiedinto subfamily 1, group H (referred to herein as “Group H nuclearreceptors”). The members of each group share 40-60% amino acid identityin the E (ligand binding) domain (Laudet et al., A Unified NomenclatureSystem for the Nuclear Receptor Subfamily, 1999; Cell 97: 161-163). Inaddition to the ecdysone receptor, other members of this nuclearreceptor subfamily 1, group H include: ubiquitous receptor (UR), Orphanreceptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1), RXRinteracting protein-15 (RIP-15), liver x receptor β (LXRβ), steroidhormone receptor like protein (RLD-1), liver x receptor (LXR), liver xreceptor α (LXRα), farnesoid x receptor (FXR), receptor interactingprotein 14 (RIP-14), and farnesol receptor (HRR-1).

To develop an improved Group H nuclear receptor-based inducible geneexpression system in which ligand binding or ligand specificity ismodified, Applicants created several substitution mutant EcRs thatcomprise substituted amino acid residues in the ligand binding domain(LBD). A homology modeling and docking approach was used to predictcritical residues that mediate binding of ecdysteroids andnon-ecdysteroids to the EcR LBD. These substitution mutant EcRs wereevaluated in ligand binding and transactivation assays. As presentedherein, Applicants' novel substitution mutant nuclear receptors andtheir use in a nuclear receptor-based inducible gene expression systemprovides an improved inducible gene expression system in bothprokaryotic and eukaryotic host cells in which ligand sensitivity andmagnitude of transactivation may be selected as desired, depending uponthe application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: In vitro ³H-RH2485 ligand binding of full-length A110P CfEcRmutant while steroid binding is completely disrupted. The ligand bindingvalues are expressed as specific counts (specific dpm).

FIG. 2: Transactivation of reporter genes through GAL4/CfEcR-A/BCDEF(full length CfEcR) or its GAL4/A110P mutant version constructstransfected into NIH3T3 cells along with VP16LmUSP-EF and pFREcRE byPonA or GST™-E. The numbers on top of the bars indicate fold increaseover DMSO levels.

FIG. 3: Transactivation of reporter genes through IE1VP16/CfEcRCDEF(Example 1.5) or its VP16/A110P mutant version constructs (Example 1.6)transfected into L57 cells along with pMK43.2 reporter by 20E or GST™-E.The numbers on top of the bars indicate fold increase over DMSO levels.

FIG. 4: Transactivation of reporter genes through GAL4/CfEcR-A/BCDEF(full length CfEcR) or its GAL4/A110 mutant versions (A110S, A110P,A110L, and A110M) constructs transfected into NIH3T3 cells along withVP16LmUSP-EF and pFREcRE by PonA or GST™-E. The numbers on top of thebars indicate fold increase over DMSO levels.

FIG. 5: In vitro ³H-PonA ligand binding of wild-type CfEcR-A/BCDEF (fulllength CfEcR) or its A110 mutant versions (A110S, A110P, A110L, andA110M). The ligand binding values are expressed as specific counts(specific dpm).

FIG. 6: In vitro ³H-RH2485 ligand binding of wild-type CfEcR-A/BCDEF(full length CfEcR) or its A110 mutant versions (A110S, A110P, A110L,and A110M). These values were expressed as specific counts (specificdpm).

DETAILED DESCRIPTION OF THE INVENTION

Applicants describe herein the construction of Group H nuclear receptorsthat comprise substitution mutations (referred to herein as“substitution mutants”) at amino acid residues that are involved inligand binding to a Group H nuclear receptor ligand binding domain thataffect the ligand sensitivity and magnitude of induction of the Group Hnuclear receptor and the demonstration that these substitution mutantnuclear receptors are useful in methods of modulating gene expression.

Specifically, Applicants have developed a novel nuclear receptor-basedinducible gene expression system comprising a Group H nuclear receptorligand binding domain comprising a substitution mutation. Applicantshave shown that the effect of such a substitution mutation may increaseor reduce ligand binding activity or ligand sensitivity and the ligandmay be steroid or non-steroid specific. Thus, Applicants' inventionprovides a Group H nuclear receptor-based inducible gene expressionsystem useful for modulating expression of a gene of interest in a hostcell. In a particularly desirable embodiment, Applicants' inventionprovides an ecdysone receptor-based inducible gene expression systemthat responds solely to either steroidal ligand or non-steroidal ligand.In addition, the present invention also provides an improvednon-steroidal ligand responsive ecdysone receptor-based inducible geneexpression system. Thus, Applicants' novel inducible gene expressionsystem and its use in methods of modulating gene expression in a hostcell overcome the limitations of currently available inducibleexpression systems and provide the skilled artisan with an effectivemeans to control gene expression.

The present invention is useful for applications such as gene therapy,large scale production of proteins and antibodies, cell-based highthroughput screening assays, orthogonal ligand screening assays,functional genomics, proteomics, metabolomics, and regulation of traitsin transgenic organisms, where control of gene expression levels isdesirable. An advantage of Applicants' invention is that it provides ameans to regulate gene expression and to tailor expression levels tosuit the user's requirements.

DEFINITIONS

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided and should be helpful inunderstanding the scope and practice of the present invention.

In a specific embodiment, the term “about” or “approximately” meanswithin 20%, preferably within 10%, more preferably within 5%, and evenmore preferably within 1% of a given value or range.

The term “substantially free” means that a composition comprising “A”(where “A” is a single protein, DNA molecule, vector, recombinant hostcell, etc.) is substantially free of “B” (where “B” comprises one ormore contaminating proteins, DNA molecules, vectors, etc.) when at leastabout 75% by weight of the proteins, DNA, vectors (depending on thecategory of species to which A and B belong) in the composition is “A”.Preferably, “A” comprises at least about 90% by weight of the A+Bspecies in the composition, most preferably at least about 99% byweight. It is also preferred that a composition, which is substantiallyfree of contamination, contain only a single molecular weight specieshaving the activity or characteristic of the species of interest.

The term “isolated” for the purposes of the present invention designatesa biological material (nucleic acid or protein) that has been removedfrom its original environment (the environment in which it is naturallypresent). For example, a polynucleotide present in the natural state ina plant or an animal is not isolated, however the same polynucleotideseparated from the adjacent nucleic acids in which it is naturallypresent, is considered “isolated”. The term “purified” does not requirethe material to be present in a form exhibiting absolute purity,exclusive of the presence of other compounds. It is rather a relativedefinition.

A polynucleotide is in the “purified” state after purification of thestarting material or of the natural material by at least one order ofmagnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

A “nucleic acid” is a polymeric compound comprised of covalently linkedsubunits called nucleotides. Nucleic acid includes polyribonucleic acid(RNA) and polydeoxyribonucleic acid (DNA), both of which may besingle-stranded or double-stranded. DNA includes but is not limited tocDNA, genomic DNA, plasmids DNA, synthetic DNA, and semi-synthetic DNA.DNA may be linear, circular, or supercoiled.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

The term “fragment” will be understood to mean a nucleotide sequence ofreduced length relative to the reference nucleic acid and comprising,over the common portion, a nucleotide sequence identical to thereference nucleic acid. Such a nucleic acid fragment according to theinvention may be, where appropriate, included in a larger polynucleotideof which it is a constituent. Such fragments comprise, or alternativelyconsist of, oligonucleotides ranging in length from at least 6, 8, 9,10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51,54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200,300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a nucleicacid according to the invention.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A “gene” refers to an assembly of nucleotides that encode a polypeptide,and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to anucleic acid fragment that expresses a specific protein or polypeptide,including regulatory sequences preceding (5′ non-coding sequences) andfollowing (3′ non-coding sequences) the coding sequence. “Native gene”refers to a gene as found in nature with its own regulatory sequences.“Chimeric gene” refers to any gene that is not a native gene, comprisingregulatory and/or coding sequences that are not found together innature. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A chimericgene may comprise coding sequences derived from different sources and/orregulatory sequences derived from different sources. “Endogenous gene”refers to a native gene in its natural location in the genome of anorganism. A “foreign” gene or “heterologous” gene refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

The term “genome” includes chromosomal as well as mitochondrial,chloroplast and viral DNA or RNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., 1989 infra). Hybridization andwashing conditions are well known and exemplified in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(1989), particularly Chapter 11 and Table 11.1 therein (entirelyincorporated herein by reference). The conditions of temperature andionic strength determine the “stringency” of the hybridization.

Stringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC,0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5%SDS). Moderate stringency hybridization conditions correspond to ahigher T_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The term “complementary” is usedto describe the relationship between nucleotide bases that are capableof hybridizing to one another. For example, with respect to DNA,adenosine is complementary to thymine and cytosine is complementary toguanine. Accordingly, the instant invention also includes isolatednucleic acid fragments that are complementary to the complete sequencesas disclosed or used herein as well as those substantially similarnucleic acid sequences.

In a specific embodiment of the invention, polynucleotides are detectedby employing hybridization conditions comprising a hybridization step atT_(m) of 55° C., and utilizing conditions as set forth above. In apreferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 63° C.; in an even more preferred embodiment,the T_(m) is 65° C.

Post-hybridization washes also determine stringency conditions. One setof preferred conditions uses a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 minutes (min), then repeated with2×SSC, 0.5% SDS at 45° C. for 30 minutes, and then repeated twice with0.2×SSC, 0.5% SDS at 50° C. for 30 minutes. A more preferred set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Anotherpreferred set of highly stringent conditions uses two final washes in0.1×SSC, 0.1% SDS at 65° C. Hybridization requires that the two nucleicacids comprise complementary sequences, although depending on thestringency of the hybridization, mismatches between bases are possible.

The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8).

In a specific embodiment of the invention, polynucleotides are detectedby employing hybridization conditions comprising a hybridization step inless than 500 mM salt and at least 37 degrees Celsius, and a washingstep in 2×SSPE at at least 63 degrees Celsius. In a preferredembodiment, the hybridization conditions comprise less than 200 mM saltand at least 37 degrees Celsius for the hybridization step. In a morepreferred embodiment, the hybridization conditions comprise 2×SSPE and63 degrees Celsius for both the hybridization and washing steps.

In one embodiment, the length for a hybridizable nucleic acid is atleast about 10 nucleotides. Preferable a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; morepreferably at least about 20 nucleotides; and most preferably the lengthis at least 30 nucleotides. Furthermore, the skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as length of theprobe.

The term “probe” refers to a single-stranded nucleic acid molecule thatcan base pair with a complementary single stranded target nucleic acidto form a double-stranded molecule.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 18 nucleotides, that is hybridizable to a genomicDNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule.Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. A labeled oligonucleotide can be used as a probe to detectthe presence of a nucleic acid. Oligonucleotides (one or both of whichmay be labeled) can be used as PCR primers, either for cloning fulllength or a fragment of a nucleic acid, or to detect the presence of anucleic acid. An oligonucleotide can also be used to form a triple helixwith a DNA molecule. Generally, oligonucleotides are preparedsynthetically, preferably on a nucleic acid synthesizer. Accordingly,oligonucleotides can be prepared with non-naturally occurringphosphoester analog bonds, such as thioester bonds, etc.

A “primer” is an oligonucleotide that hybridizes to a target nucleicacid sequence to create a double stranded nucleic acid region that canserve as an initiation point for DNA synthesis under suitableconditions. Such primers may be used in a polymerase chain reaction.

“Polymerase chain reaction” is abbreviated PCR and means an in vitromethod for enzymatically amplifying specific nucleic acid sequences. PCRinvolves a repetitive series of temperature cycles with each cyclecomprising three stages: denaturation of the template nucleic acid toseparate the strands of the target molecule, annealing a single strandedPCR oligonucleotide primer to the template nucleic acid, and extensionof the annealed primer(s) by DNA polymerase. PCR provides a means todetect the presence of the target molecule and, under quantitative orsemi-quantitative conditions, to determine the relative amount of thattarget molecule within the starting pool of nucleic acids.

“Reverse transcription-polymerase chain reaction” is abbreviated RT-PCRand means an in vitro method for enzymatically producing a target cDNAmolecule or molecules from an RNA molecule or molecules, followed byenzymatic amplification of a specific nucleic acid sequence or sequenceswithin the target cDNA molecule or molecules as described above. RT-PCRalso provides a means to detect the presence of the target molecule and,under quantitative or semi-quantitative conditions, to determine therelative amount of that target molecule within the starting pool ofnucleic acids.

A DNA “coding sequence” is a double-stranded DNA sequence that istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.“Suitable regulatory sequences” refer to nucleotide sequences locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing site, effector binding site and stem-loop structure. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and evensynthetic DNA sequences. If the coding sequence is intended forexpression in a eukaryotic cell, a polyadenylation signal andtranscription termination sequence will usually be located 3′ to thecoding sequence.

“Open reading frame” is abbreviated ORF and means a length of nucleicacid sequence, either DNA, cDNA or RNA, that comprises a translationstart signal or initiation codon, such as an ATG or AUG, and atermination codon and can be potentially translated into a polypeptidesequence.

The term “head-to-head” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-head orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 5′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds away from the 5′ end ofthe other polynucleotide. The term “head-to-head” may be abbreviated(5′)-to-(5′) and may also be indicated by the symbols (←→) or(3′←5′5′→3′).

The term “tail-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a tail-to-tail orientation when the 3′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds toward the otherpolynucleotide. The term “tail-to-tail” may be abbreviated (3′)-to-(3′)and may also be indicated by the symbols (→←) or (5′→3′3′←5′).

The term “head-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-tail orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds in the same directionas that of the other polynucleotide. The term “head-to-tail” may beabbreviated (5′)-to-(3′) and may also be indicated by the symbols (→→)or (5′→3′5′→3′).

The term “downstream” refers to a nucleotide sequence that is located 3′to reference nucleotide sequence. In particular, downstream nucleotidesequences generally relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to reference nucleotide sequence. In particular, upstream nucleotidesequences generally relate to sequences that are located on the 5′ sideof a coding sequence or starting point of transcription. For example,most promoters are located upstream of the start site of transcription.

The terms “restriction endonuclease” and “restriction enzyme” refer toan enzyme that binds and cuts within a specific nucleotide sequencewithin double stranded DNA.

“Homologous recombination” refers to the insertion of a foreign DNAsequence into another DNA molecule, e.g., insertion of a vector in achromosome. Preferably, the vector targets a specific chromosomal sitefor homologous recombination. For specific homologous recombination, thevector will contain sufficiently long regions of homology to sequencesof the chromosome to allow complementary binding and incorporation ofthe vector into the chromosome. Longer regions of homology, and greaterdegrees of sequence similarity, may increase the efficiency ofhomologous recombination.

Several methods known in the art may be used to propagate apolynucleotide according to the invention. Once a suitable host systemand growth conditions are established, recombinant expression vectorscan be propagated and prepared in quantity. As described herein, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

A “vector” is any means for the cloning of and/or transfer of a nucleicacid into a host cell. A vector may be a replicon to which another DNAsegment may be attached so as to bring about the replication of theattached segment. A “replicon” is any genetic element (e.g., plasmid,phage, cosmid, chromosome, virus) that functions as an autonomous unitof DNA replication in vivo, i.e., capable of replication under its owncontrol. The term “vector” includes both viral and nonviral means forintroducing the nucleic acid into a cell in vitro, ex vivo or in vivo. Alarge number of vectors known in the art may be used to manipulatenucleic acids, incorporate response elements and promoters into genes,etc. Possible vectors include, for example, plasmids or modified virusesincluding, for example bacteriophages such as lambda derivatives, orplasmids such as pBR322 or pUC plasmid derivatives, or the Bluescriptvector. For example, the insertion of the DNA fragments corresponding toresponse elements and promoters into a suitable vector can beaccomplished by ligating the appropriate DNA fragments into a chosenvector that has complementary cohesive termini. Alternatively, the endsof the DNA molecules may be enzymatically modified or any site may beproduced by ligating nucleotide sequences (linkers) into the DNAtermini. Such vectors may be engineered to contain selectable markergenes that provide for the selection of cells that have incorporated themarker into the cellular genome. Such markers allow identificationand/or selection of host cells that incorporate and express the proteinsencoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in awide variety of gene delivery applications in cells, as well as livinganimal subjects. Viral vectors that can be used include but are notlimited to retrovirus, adeno-associated virus, pox, baculovirus,vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, andcaulimovirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers. In addition to a nucleic acid, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(transfer to which tissues, duration of expression, etc.).

The term “plasmid” refers to an extra chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A “cloning vector” is a “replicon”, which is a unit length of a nucleicacid, preferably DNA, that replicates sequentially and which comprisesan origin of replication, such as a plasmid, phage or cosmid, to whichanother nucleic acid segment may be attached so as to bring about thereplication of the attached segment. Cloning vectors may be capable ofreplication in one cell type and expression in another (“shuttlevector”).

Vectors may be introduced into the desired host cells by methods knownin the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; and Hartmutet al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

A polynucleotide according to the invention can also be introduced invivo by lipofection. For the past decade, there has been increasing useof liposomes for encapsulation and transfection of nucleic acids invitro. Synthetic cationic lipids designed to limit the difficulties anddangers encountered with liposome-mediated transfection can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Felgner et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84: 7413; Mackey,et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031; and Ulmer etal., 1993, Science 259: 1745-1748). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Feigner andRingold, 1989, Science 337:387-388). Particularly useful lipid compoundsand compositions for transfer of nucleic acids are described inInternational Patent Publications WO95/18863 and WO96/17823, and in U.S.Pat. No. 5,459,127. The use of lipofection to introduce exogenous genesinto the specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. It is clear that directing transfection to particular celltypes would be particularly preferred in a tissue with cellularheterogeneity, such as pancreas, liver, kidney, and the brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting (Mackey, et al., 1988, supra). Targeted peptides, e.g.,hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as a naked DNA plasmid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., 1992, Hum. Gene Ther. 3: 147-154; and Wu and Wu, 1987, J. Biol.Chem. 262: 4429-4432).

The term “transfection” means the uptake of exogenous or heterologousRNA or DNA by a cell. A cell has been “transfected” by exogenous orheterologous RNA or DNA when such RNA or DNA has been introduced insidethe cell. A cell has been “transformed” by exogenous or heterologous RNAor DNA when the transfected RNA or DNA effects a phenotypic change. Thetransforming RNA or DNA can be integrated (covalently linked) intochromosomal DNA making up the genome of the cell.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The term “genetic region” will refer to a region of a nucleic acidmolecule or a nucleotide sequence that comprises a gene encoding apolypeptide.

In addition, the recombinant vector comprising a polynucleotideaccording to the invention may include one or more origins forreplication in the cellular hosts in which their amplification or theirexpression is sought, markers or selectable markers.

The term “selectable marker” means an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,resistance to a herbicide, colorimetric markers, enzymes, fluorescentmarkers, and the like, wherein the effect is used to track theinheritance of a nucleic acid of interest and/or to identify a cell ororganism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like.

The term “reporter gene” means a nucleic acid encoding an identifyingfactor that is able to be identified based upon the reporter gene'seffect, wherein the effect is used to track the inheritance of a nucleicacid of interest, to identify a cell or organism that has inherited thenucleic acid of interest, and/or to measure gene expression induction ortranscription. Examples of reporter genes known and used in the artinclude: luciferase (Luc), green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable marker genes may also beconsidered reporter genes.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. Promotersthat cause a gene to be expressed in a specific cell type are commonlyreferred to as “cell-specific promoters” or “tissue-specific promoters”.Promoters that cause a gene to be expressed at a specific stage ofdevelopment or cell differentiation are commonly referred to as“developmentally-specific promoters” or “cell differentiation-specificpromoters”. Promoters that are induced and cause a gene to be expressedfollowing exposure or treatment of the cell with an agent, biologicalmolecule, chemical, ligand, light, or the like that induces the promoterare commonly referred to as “inducible promoters” or “regulatablepromoters”. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical promoter activity.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced (if the coding sequence contains introns) and translated intothe protein encoded by the coding sequence.

“Transcriptional and translational control sequences” are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

The term “response element” means one or more cis-acting DNA elementswhich confer responsiveness on a promoter mediated through interactionwith the DNA-binding domains of the first chimeric gene. This DNAelement may be either palindromic (perfect or imperfect) in its sequenceor composed of sequence motifs or half sites separated by a variablenumber of nucleotides. The half sites can be similar or identical andarranged as either direct or inverted repeats or as a single half siteor multimers of adjacent half sites in tandem. The response element maycomprise a minimal promoter isolated from different organisms dependingupon the nature of the cell or organism into which the response elementwill be incorporated. The DNA binding domain of the first hybrid proteinbinds, in the presence or absence of a ligand, to the DNA sequence of aresponse element to initiate or suppress transcription of downstreamgene(s) under the regulation of this response element. Examples of DNAsequences for response elements of the natural ecdysone receptorinclude: RRGG/TTCANTGAC/ACYY (see Cherbas L., et. al., (1991), GenesDev. 5, 120-131); AGGTCAN_((n))AGGTCA, where N_((n)) can be one or morespacer nucleotides (see D'Avino P P., et. al., (1995), Mol. CellEndocrinol, 113, 1-9); and GGGTTGAATGAATTT (see Antoniewski C., et. al.,(1994). Mol. Cell Biol. 14, 4465-4474).

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from anucleic acid or polynucleotide. Expression may also refer to translationof mRNA into a protein or polypeptide.

The terms “cassette”, “expression cassette” and “gene expressioncassette” refer to a segment of DNA that can be inserted into a nucleicacid or polynucleotide at specific restriction sites or by homologousrecombination. The segment of DNA comprises a polynucleotide thatencodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. “Transformationcassette” refers to a specific vector comprising a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.Cassettes, expression cassettes, gene expression cassettes andtransformation cassettes of the invention may also comprise elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

For purposes of this invention, the term “gene switch” refers to thecombination of a response element associated with a promoter, and an EcRbased system which, in the presence of one or more ligands, modulatesthe expression of a gene into which the response element and promoterare incorporated.

The terms “modulate” and “modulates” mean to induce, reduce or inhibitnucleic acid or gene expression, resulting in the respective induction,reduction or inhibition of protein or polypeptide production.

The plasmids or vectors according to the invention may further compriseat least one promoter suitable for driving expression of a gene in ahost cell. The term “expression vector” means a vector, plasmid orvehicle designed to enable the expression of an inserted nucleic acidsequence following transformation into the host. The cloned gene, i.e.,the inserted nucleic acid sequence, is usually placed under the controlof control elements such as a promoter, a minimal promoter, an enhancer,or the like. Initiation control regions or promoters, which are usefulto drive expression of a nucleic acid in the desired host cell arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to: viral promoters, bacterialpromoters, animal promoters, mammalian promoters, synthetic promoters,constitutive promoters, tissue specific promoter, developmental specificpromoters, inducible promoters, light regulated promoters; CYC1, HIS3,GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TPI, alkaline phosphatase promoters (useful for expression inSaccharomyces); AOX1 promoter (useful for expression in Pichia);β-lactamase, lac, ara, tet, trp, lP_(L), lP_(R), T7, tac, and trcpromoters (useful for expression in Escherichia coli); light regulated-,seed specific-, pollen specific-, ovary specific-, pathogenesis ordisease related-, cauliflower mosaic virus 35S, CMV 35S minimal, cassayavein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose1,5-bisphosphate carboxylase, shoot-specific, root specific, chitinase,stress inducible, rice tungro bacilliform virus, plant super-promoter,potato leucine aminopeptidase, nitrate reductase, mannopine synthase,nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters(useful for expression in plant cells); animal and mammalian promotersknown in the art include, but are not limited to, the SV40 early (SV40e)promoter region, the promoter contained in the 3′ long terminal repeat(LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or majorlate promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus(CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase(TK) promoter, a baculovirus 1E1 promoter, an elongation factor 1 alpha(EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin(Ubc) promoter, an albumin promoter, the regulatory sequences of themouse metallothionein-L promoter and transcriptional control regions,the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and thelike), the promoters of the intermediate filaments (desmin,neurofilaments, keratin, GFAP, and the like), the promoters oftherapeutic genes (of the MDR, CFTR or factor VIII type, and the like),pathogenesis or disease related-promoters, and promoters that exhibittissue specificity and have been utilized in transgenic animals, such asthe elastase I gene control region which is active in pancreatic acinarcells; insulin gene control region active in pancreatic beta cells,immunoglobulin gene control region active in lymphoid cells, mousemammary tumor virus control region active in testicular, breast,lymphoid and mast cells; albumin gene, Apo AI and Apo AII controlregions active in liver, alpha-fetoprotein gene control region active inliver, alpha 1-antitrypsin gene control region active in the liver,beta-globin gene control region active in myeloid cells, myelin basicprotein gene control region active in oligodendrocyte cells in thebrain, myosin light chain-2 gene control region active in skeletalmuscle, and gonadotropic releasing hormone gene control region active inthe hypothalamus, pyruvate kinase promoter, villin promoter, promoter ofthe fatty acid binding intestinal protein, promoter of the smooth musclecell α-actin, and the like. In addition, these expression sequences maybe modified by addition of enhancer or regulatory sequences and thelike.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor 1 (EF1) enhancer, yeast enhancers, viral geneenhancers, and the like.

Termination control regions, i.e., terminator or polyadenylationsequences, may also be derived from various genes native to thepreferred hosts. Optionally, a termination site may be unnecessary,however, it is most preferred if included. In a preferred embodiment ofthe invention, the termination control region may be comprise or bederived from a synthetic sequence, synthetic polyadenylation signal, anSV40 late polyadenylation signal, an SV40 polyadenylation signal, abovine growth hormone (BGH) polyadenylation signal, viral terminatorsequences, or the like.

The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)”refer to DNA sequences located downstream (3′) of a coding sequence andmay comprise polyadenylation [poly(A)] recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor.

“Regulatory region” means a nucleic acid sequence that regulates theexpression of a second nucleic acid sequence. A regulatory region mayinclude sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin that are responsible for expressing differentproteins or even synthetic proteins (a heterologous region). Inparticular, the sequences can be sequences of prokaryotic, eukaryotic,or viral genes or derived sequences that stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, promoters, enhancers, transcriptionaltermination sequences, and signal sequences which direct the polypeptideinto the secretory pathways of the target cell.

A regulatory region from a “heterologous source” is a regulatory regionthat is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but which are designed by one having ordinary skill inthe art.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or thecoding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA,or other RNA that is not translated yet has an effect on cellularprocesses.

A “polypeptide” is a polymeric compound comprised of covalently linkedamino acid residues. Amino acids have the following general structure:

Amino acids are classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup. A polypeptide of the invention preferably comprises at leastabout 14 amino acids.

A “protein” is a polypeptide that performs a structural or functionalrole in a living cell.

An “isolated polypeptide” or “isolated protein” is a polypeptide orprotein that is substantially free of those compounds that are normallyassociated therewith in its natural state (e.g., other proteins orpolypeptides, nucleic acids, carbohydrates, lipids). “Isolated” is notmeant to exclude artificial or synthetic mixtures with other compounds,or the presence of impurities which do not interfere with biologicalactivity, and which may be present, for example, due to incompletepurification, addition of stabilizers, or compounding into apharmaceutically acceptable preparation.

A “substitution mutant polypeptide” or a “substitution mutant” will beunderstood to mean a mutant polypeptide comprising a substitution of atleast one (1) wild-type or naturally occurring amino acid with adifferent amino acid relative to the wild-type or naturally occurringpolypeptide. A substitution mutant polypeptide may comprise only one (1)wild-type or naturally occurring amino acid substitution and may bereferred to as a “point mutant” or a “single point mutant” polypeptide.Alternatively, a substitution mutant polypeptide may comprise asubstitution of two (2) or more wild-type or naturally occurring aminoacids with 2 or more amino acids relative to the wild-type or naturallyoccurring polypeptide. According to the invention, a Group H nuclearreceptor ligand binding domain polypeptide comprising a substitutionmutation comprises a substitution of at least one (1) wild-type ornaturally occurring amino acid with a different amino acid relative tothe wild-type or naturally occurring Group H nuclear receptor ligandbinding domain polypeptide.

Wherein the substitution mutant polypeptide comprises a substitution oftwo (2) or more wild-type or naturally occurring amino acids, thissubstitution may comprise either an equivalent number of wild-type ornaturally occurring amino acids deleted for the substitution, i.e., 2wild-type or naturally occurring amino acids replaced with 2non-wild-type or non-naturally occurring amino acids, or anon-equivalent number of wild-type amino acids deleted for thesubstitution, i.e., 2 wild-type amino acids replaced with 1non-wild-type amino acid (a substitution+deletion mutation), or 2wild-type amino acids replaced with 3 non-wild-type amino acids (asubstitution+insertion mutation). Substitution mutants may be describedusing an abbreviated nomenclature system to indicate the amino acidresidue and number replaced within the reference polypeptide sequenceand the new substituted amino acid residue. For example, a substitutionmutant in which the twentieth (20^(th)) amino acid residue of apolypeptide is substituted may be abbreviated as “x20z”, wherein “x” isthe amino acid to be replaced, “20” is the amino acid residue positionor number within the polypeptide, and “z” is the new substituted aminoacid. Therefore, a substitution mutant abbreviated interchangeably as“E20A” or “Glu20Ala” indicates that the mutant comprises an alanineresidue (commonly abbreviated in the art as “A” or “Ala”) in place ofthe glutamic acid (commonly abbreviated in the art as “E” or “Glu”) atposition 20 of the polypeptide.

A substitution mutation may be made by any technique for mutagenesisknown in the art, including but not limited to, in vitro site-directedmutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem. 253: 6551;Zoller and Smith, 1984, DNA 3: 479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 710),use of TAB® linkers (Pharmacia), restriction endonucleasedigestion/fragment deletion and substitution,PCR-mediated/oligonucleotide-directed mutagenesis, and the like.PCR-based techniques are preferred for site-directed mutagenesis (seeHiguchi, 1989, “Using PCR to Engineer DNA”, in PCR Technology:Principles and Applications for DNA Amplcation, H. Erlich, ed., StocktonPress, Chapter 6, pp. 61-70).

“Fragment” of a polypeptide according to the invention will beunderstood to mean a polypeptide whose amino acid sequence is shorterthan that of the reference polypeptide and which comprises, over theentire portion with these reference polypeptides, an identical aminoacid sequence. Such fragments may, where appropriate, be included in alarger polypeptide of which they are a part. Such fragments of apolypeptide according to the invention may have a length of at least 2,3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30,35, 40, 45, 50, 100, 200, 240, or 300 amino acids.

A “variant” of a polypeptide or protein is any analogue, fragment,derivative, or mutant which is derived from a polypeptide or protein andwhich retains at least one biological property of the polypeptide orprotein. Different variants of the polypeptide or protein may exist innature. These variants may be allelic variations characterized bydifferences in the nucleotide sequences of the structural gene codingfor the protein, or may involve differential splicing orpost-translational modification. The skilled artisan can producevariants having single or multiple amino acid substitutions, deletions,additions, or replacements. These variants may include, inter alia: (a)variants in which one or more amino acid residues are substituted withconservative or non-conservative amino acids, (b) variants in which oneor more amino acids are added to the polypeptide or protein, (c)variants in which one or more of the amino acids includes a substituentgroup, and (d) variants in which the polypeptide or protein is fusedwith another polypeptide such as serum albumin. The techniques forobtaining these variants, including genetic (suppressions, deletions,mutations, etc.), chemical, and enzymatic techniques, are known topersons having ordinary skill in the art. A variant polypeptidepreferably comprises at least about 14 amino acids.

A “heterologous protein” refers to a protein not naturally produced inthe cell.

A “mature protein” refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present. Pre- and propeptides may be but are notlimited to intracellular localization signals.

The term “signal peptide” refers to an amino terminal polypeptidepreceding the secreted mature protein. The signal peptide is cleavedfrom and is therefore not present in the mature protein. Signal peptideshave the function of directing and translocating secreted proteinsacross cell membranes. Signal peptide is also referred to as signalprotein.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide, thatdirects the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

The term “homology” refers to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown to the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions that form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s) and size determination of thedigested fragments.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., 1987, Cell 50: 667.). Such proteins (and their encoding genes)have sequence homology, as reflected by their high degree of sequencesimilarity. However, in common usage and in the instant application, theterm “homologous,” when modified with an adverb such as “highly,” mayrefer to sequence similarity and not a common evolutionary origin.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., 1987, Cell 50:667).

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Sambrook et al., 1989, supra.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotide bases that donot substantially affect the functional properties of the resultingtranscript. It is therefore understood that the invention encompassesmore than the specific exemplary sequences. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Moreover, the skilled artisan recognizes that substantially similarsequences encompassed by this invention are also defined by theirability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65°C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), withthe sequences exemplified herein. Substantially similar nucleic acidfragments of the instant invention are those nucleic acid fragmentswhose DNA sequences are at least 70% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Preferred substantiallynucleic acid fragments of the instant invention are those nucleic acidfragments whose DNA sequences are at least 80% identical to the DNAsequence of the nucleic acid fragments reported herein. More preferrednucleic acid fragments are at least 90% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Even more preferred arenucleic acid fragments that are at least 95% identical to the DNAsequence of the nucleic acid fragments reported herein.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than about 40% of the amino acidsare identical, or greater than 60% are similar (functionally identical).Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993) J. Mol. Biol. 215: 403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesmay be performed using the Clustal method of alignment (Higgins andSharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method may be selected: KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include but is not limited to the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215: 403-410(1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715USA). Within the context of this application it will be understood thatwhere sequence analysis software is used for analysis, that the resultsof the analysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters which originally load with thesoftware when first initialized.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

As used herein, two or more individually operable gene regulationsystems are said to be “orthogonal” when; a) modulation of each of thegiven systems by its respective ligand, at a chosen concentration,results in a measurable change in the magnitude of expression of thegene of that system, and b) the change is statistically significantlydifferent than the change in expression of all other systemssimultaneously operable in the cell, tissue, or organism, regardless ofthe simultaneity or sequentially of the actual modulation. Preferably,modulation of each individually operable gene regulation system effectsa change in gene expression at least 2-fold greater than all otheroperable systems in the cell, tissue, or organism. More preferably, thechange is at least 5-fold greater. Even more preferably, the change isat least 10-fold greater. Still more preferably, the change is at least100 fold greater. Even still more preferably, the change is at least500-fold greater. Ideally, modulation of each of the given systems byits respective ligand at a chosen concentration results in a measurablechange in the magnitude of expression of the gene of that system and nomeasurable change in expression of all other systems operable in thecell, tissue, or organism. In such cases the multiple inducible generegulation system is said to be “fully orthogonal”. The presentinvention is useful to search for orthogonal ligands and orthogonalreceptor-based gene expression systems such as those described inco-pending U.S. application 60/237,446, which is incorporated herein byreference in its entirety.

Gene Expression Modulation System of the Invention

Applicants have identified herein amino acid residues that are involvedin ligand binding to a Group H nuclear receptor ligand binding domainthat affect the ligand sensitivity and magnitude of induction in anecdysone receptor-based inducible gene expression system. Applicantsdescribe herein the construction of Group H nuclear receptors thatcomprise substitution mutations (referred to herein as “substitutionmutants”) at these critical residues and the demonstration that thesesubstitution mutant nuclear receptors are useful in methods ofmodulating gene expression. As presented herein, Applicants' novelsubstitution mutant nuclear receptors and their use in a nuclearreceptor-based inducible gene expression system provides an improvedinducible gene expression system in both prokaryotic and eukaryotic hostcells in which ligand sensitivity and magnitude of transactivation maybe selected as desired, depending upon the application.

Thus, the present invention relates to novel substitution mutant Group Hnuclear receptor polynucleotides and polypeptides, a nuclearreceptor-based inducible gene expression system comprising such mutatedGroup H nuclear receptor polynucleotides and polypeptides, and methodsof modulating the expression of a gene within a host cell using such anuclear receptor-based inducible gene expression system.

In particular, the present invention relates to a gene expressionmodulation system comprising at least one gene expression cassette thatis capable of being expressed in a host cell comprising a polynucleotidethat encodes a polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation. Preferably, the GroupH nuclear receptor ligand binding domain comprising a substitutionmutation is from an ecdysone receptor, a ubiquitous receptor, an orphanreceptor 1, a NER-1, a steroid hormone nuclear receptor 1, a retinoid Xreceptor interacting protein −15, a liver X receptor β, a steroidhormone receptor like protein, a liver X receptor, a liver X receptor α,a farnesoid X receptor, a receptor interacting protein 14, and afarnesol receptor. More preferably, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is from an ecdysonereceptor.

In a specific embodiment, the gene expression modulation systemcomprises a gene expression cassette comprising a polynucleotide thatencodes a polypeptide comprising a transactivation domain, a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation. The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another specific embodiment, the gene expression modulation systemcomprises a gene expression cassette comprising a) a polynucleotide thatencodes a polypeptide comprising a transactivation domain, a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, and b) a secondnuclear receptor ligand binding domain selected from the groupconsisting of a vertebrate retinoid X receptor ligand binding domain, aninvertebrate retinoid X receptor ligand binding domain, an ultraspiracleprotein ligand binding domain, and a chimeric ligand binding domaincomprising two polypeptide fragments, wherein the first polypeptidefragment is from a vertebrate retinoid X receptor ligand binding domain,an invertebrate retinoid X receptor ligand binding domain, or anultraspiracle protein ligand binding domain, and the second polypeptidefragment is from a different vertebrate retinoid X receptor ligandbinding domain, invertebrate retinoid X receptor ligand binding domain,or ultraspiracle protein ligand binding domain. The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another specific embodiment, the gene expression modulation systemcomprises a first gene expression cassette comprising a polynucleotidethat encodes a first polypeptide comprising a DNA-binding domain thatrecognizes a response element associated with a gene whose expression isto be modulated and a nuclear receptor ligand binding domain, and asecond gene expression cassette comprising a polynucleotide that encodesa second polypeptide comprising a transactivation domain and a nuclearreceptor ligand binding domain, wherein one of the nuclear receptorligand binding domains is a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation. In a preferred embodiment,the first polypeptide is substantially free of a transactivation domainand the second polypeptide is substantially free of a DNA bindingdomain. For purposes of the invention, “substantially free” means thatthe protein in question does not contain a sufficient sequence of thedomain in question to provide activation or binding activity. The geneexpression modulation system may further comprise a third geneexpression cassette comprising: i) a response element recognized by theDNA-binding domain of the first polypeptide of the first gene expressioncassette; ii) a promoter that is activated by the transactivation domainof the second polypeptide of the second gene expression cassette; andiii) a gene whose expression is to be modulated.

Wherein when only one nuclear receptor ligand binding domain is a GroupH ligand binding domain comprising a substitution mutation, the othernuclear receptor ligand binding domain may be from any other nuclearreceptor that forms a dimer with the Group H ligand binding domaincomprising the substitution mutation. For example, when the Group Hnuclear receptor ligand binding domain comprising a substitutionmutation is an ecdysone receptor ligand binding domain comprising asubstitution mutation, the other nuclear receptor ligand binding domain(“partner”) may be from an ecdysone receptor, a vertebrate retinoid Xreceptor (RXR), an invertebrate RXR, an ultraspiracle protein (USP), ora chimeric nuclear receptor comprising at least two different nuclearreceptor ligand binding domain polypeptide fragments selected from thegroup consisting of a vertebrate RXR, an invertebrate RXR, and a USP(see co-pending applications PCT/US01/09050, U.S. 60/294,814, and U.S.60/294,819, incorporated herein by reference in their entirety). The“partner” nuclear receptor ligand binding domain may further comprise atruncation mutation, a deletion mutation, a substitution mutation, oranother modification.

Preferably, the vertebrate RXR ligand binding domain is from a humanHomo sapiens, mouse Mus musculus, rat Rattus norvegicus, chicken Gallusgallus, pig Sus scrofa domestica, frog Xenopus laevis, zebrafish Daniorerio, tunicate Polyandrocarpa misakiensis, or jellyfish Tripedaliacysophora RXR.

Preferably, the invertebrate RXR ligand binding domain is from a locustLocusta migratoria ultraspiracle polypeptide (“LmUSP”), an ixodid tickAmblyomma americanum RXR homolog 1 (“AmaRXR1”), a ixodid tick Amblyommaamericanum RXR homolog 2 (“AmaRXR2”), a fiddler crab Celuca pugilatorRXR homolog (“CpRXR”), a beetle Tenebrio molitor RXR homolog (“TmRXR”),a honeybee Apis mellifera RXR homolog (“AmRXR”), an aphid Myzus persicaeRXR homolog (“MpRXR”), or a non-Dipteran/non-Lepidopteran RXR homolog.

Preferably, the chimeric RXR ligand binding domain comprises at leasttwo polypeptide fragments selected from the group consisting of avertebrate species RXR polypeptide fragment, an invertebrate species RXRpolypeptide fragment, and a non-Dipteran/non-Lepidopteran invertebratespecies RXR homolog polypeptide fragment. A chimeric RXR ligand bindingdomain for use in the present invention may comprise at least twodifferent species RXR polypeptide fragments, or when the species is thesame, the two or more polypeptide fragments may be from two or moredifferent isoforms of the species RXR polypeptide fragment.

In a preferred embodiment, the chimeric RXR ligand binding domaincomprises at least one vertebrate species RXR polypeptide fragment andone invertebrate species RXR polypeptide fragment.

In a more preferred embodiment, the chimeric RXR ligand binding domaincomprises at least one vertebrate species RXR polypeptide fragment andone non-Dipteran/non-Lepidopteran invertebrate species RXR homologpolypeptide fragment.

In a specific embodiment, the gene whose expression is to be modulatedis a homologous gene with respect to the host cell. In another specificembodiment, the gene whose expression is to be modulated is aheterologous gene with respect to the host cell.

The ligands for use in the present invention as described below, whencombined with the ligand binding domain of the nuclear receptor(s),which in turn are bound to the response element linked to a gene,provide the means for external temporal regulation of expression of thegene. The binding mechanism or the order in which the various componentsof this invention bind to each other, that is, for example, ligand toligand binding domain, DNA-binding domain to response element,transactivation domain to promoter, etc., is not critical.

In a specific example, binding of the ligand to the ligand bindingdomain of a Group H nuclear receptor and its nuclear receptor ligandbinding domain partner enables expression or suppression of the gene.This mechanism does not exclude the potential for ligand binding to theGroup H nuclear receptor (GHNR) or its partner, and the resultingformation of active homodimer complexes (e.g. GHNR+GHNR orpartner+partner). Preferably, one or more of the receptor domains isvaried producing a hybrid gene switch. Typically, one or more of thethree domains, DBD, LBD, and transactivation domain, may be chosen froma source different than the source of the other domains so that thehybrid genes and the resulting hybrid proteins are optimized in thechosen host cell or organism for transactivating activity, complementarybinding of the ligand, and recognition of a specific response element.In addition, the response element itself can be modified or substitutedwith response elements for other DNA binding protein domains such as theGAL-4 protein from yeast (see Sadowski, et al. (1988), Nature, 335:563-564) or LexA protein from Escherichia coli (see Brent and Ptashne(1985), Cell, 43: 729-736), or synthetic response elements specific fortargeted interactions with proteins designed, modified, and selected forsuch specific interactions (see, for example, Kim, et al. (1997), Proc.Natl. Acad. Sci., USA, 94:3 616-3620) to accommodate hybrid receptors.Another advantage of two-hybrid systems is that they allow choice of apromoter used to drive the gene expression according to a desired endresult. Such double control can be particularly important in areas ofgene therapy, especially when cytotoxic proteins are produced, becauseboth the timing of expression as well as the cells wherein expressionoccurs can be controlled. When genes, operably linked to a suitablepromoter, are introduced into the cells of the subject, expression ofthe exogenous genes is controlled by the presence of the system of thisinvention. Promoters may be constitutively or inducibly regulated or maybe tissue-specific (that is, expressed only in a particular type ofcells) or specific to certain developmental stages of the organism.

The ecdysone receptor is a member of the nuclear receptor superfamilyand classified into subfamily 1, group H (referred to herein as “Group Hnuclear receptors”). The members of each group share 40-60% amino acididentity in the E (ligand binding) domain (Laudet et al., A UnifiedNomenclature System for the Nuclear Receptor Subfamily, 1999; Cell 97:161-163). In addition to the ecdysone receptor, other members of thisnuclear receptor subfamily 1, group H include: ubiquitous receptor (UR),orphan receptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1),retinoid X receptor interacting protein −15 (RIP-15), liver X receptor β(LXRβ), steroid hormone receptor like protein (RLD-1), liver X receptor(LXR), liver X receptor α (LXRα), farnesoid X receptor (FXR), receptorinteracting protein 14 (RIP-14), and farnesol receptor (HRR-1).

Applicants have developed a CfEcR homology model and have used thishomology model together with a published Chironomous tetans ecdysonereceptor (“CtEcR”) homology model (Wurtz et al., 2000) to identifycritical residues involved in binding to steroids and non-steroids. Thesynthetic non-steroids, diacylhydrazines, have been shown to bindlepidopteran EcRs with high affinity and induce precocious incompletemolt in these insects (Wing et al., 1988) and several of these compoundsare currently marketed as insecticides. The ligand binding cavity or“pocket” of EcRs has evolved to fit the long backbone structures ofecdysteroids such as 20-hydroxyecdysone (20E). The diacylhydrazines havea compact structure compared to steroids and occupy only the bottom partof the EcR binding pocket. This leaves a few critical residues at thetop part of the binding pocket that make contact with steroids but notwith non-steroids such as bisacylhydrazines. Applicants describe hereinthe construction of mutant ecdysone receptors comprising a substitutionmutation at these binding pocket residues and have identified severalclasses of substitution mutant ecdysone receptors with modified ligandbinding and transactivation characteristics.

Given the close relatedness of ecdysone receptor to other Group Hnuclear receptors, Applicants' identified ecdysone receptor ligandbinding domain substitution mutations are also expected to work whenintroduced into the analogous position of the ligand binding domains ofother Group H nuclear receptors to modify their ligand binding or ligandsensitivity. Applicants' novel substitution mutated Group H nuclearreceptor polynucleotides and polypeptides are useful in a nuclearreceptor-based inducible gene modulation system for various applicationsincluding gene therapy, expression of proteins of interest in hostcells, production of transgenic organisms, and cell-based assays.

In particular, Applicants describe herein a novel gene expressionmodulation system comprising a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation. This gene expression systemmay be a “single switch”-based gene expression system in which thetransactivation domain, DNA-binding domain and ligand binding domain areon one encoded polypeptide. Alternatively, the gene expressionmodulation system may be a “dual switch”- or “two-hybrid”-based geneexpression modulation system in which the transactivation domain andDNA-binding domain are located on two different encoded polypeptides.Applicants have demonstrated for the first time that a substitutionmutated nuclear receptor can be used as a component of a nuclearreceptor-based inducible gene expression system to modify ligand bindingactivity and/or ligand specificity in both prokaryotic and eukaryoticcells. As discussed herein, Applicants' findings are both unexpected andsurprising.

An ecdysone receptor-based gene expression modulation system of thepresent invention may be either heterodimeric and homodimeric. Afunctional EcR complex generally refers to a heterodimeric proteincomplex consisting of two members of the steroid receptor family, anecdysone receptor protein obtained from various insects, and anultraspiracle (USP) protein or the vertebrate homolog of USP, retinoid Xreceptor protein (see Yao, et al. (1993) Nature 366: 476-479; Yao, etal., (1992) Cell 71: 63-72). However, the complex may also be ahomodimer as detailed below. The functional ecdysteroid receptor complexmay also include additional protein(s) such as immunophilins. Additionalmembers of the steroid receptor family of proteins, known astranscriptional factors (such as DHR38 or betaFTZ-1), may also be liganddependent or independent partners for EcR, USP, and/or RXR.Additionally, other cofactors may be required such as proteins generallyknown as coactivators (also termed adapters or mediators). Theseproteins do not bind sequence-specifically to DNA and are not involvedin basal transcription. They may exert their effect on transcriptionactivation through various mechanisms, including stimulation ofDNA-binding of activators, by affecting chromatin structure, or bymediating activator-initiation complex interactions. Examples of suchcoactivators include RIP140, TIF1, RAP46/Bag-1, ARA70, SRC-1/NCoA-1,TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the promiscuouscoactivator C response element B binding protein, CBP/p300 (for reviewsee Glass et al., Curr. Opin. Cell Biol. 9: 222-232, 1997). Also,protein cofactors generally known as corepressors (also known asrepressors, silencers, or silencing mediators) may be required toeffectively inhibit transcriptional activation in the absence of ligand.These corepressors may interact with the unliganded ecdysone receptor tosilence the activity at the response element. Current evidence suggeststhat the binding of ligand changes the conformation of the receptor,which results in release of the corepressor and recruitment of the abovedescribed coactivators, thereby abolishing their silencing activity.Examples of corepressors include N-CoR and SMRT (for review, see Horwitzet al. Mol Endocrinol. 10: 1167-1177, 1996). These cofactors may eitherbe endogenous within the cell or organism, or may be added exogenouslyas transgenes to be expressed in either a regulated or unregulatedfashion. Homodimer complexes of the ecdysone receptor protein, USP, orRXR may also be functional under some circumstances.

The ecdysone receptor complex typically includes proteins that aremembers of the nuclear receptor superfamily wherein all members aregenerally characterized by the presence of an amino-terminaltransactivation domain, a DNA binding domain (“DBD”), and a ligandbinding domain (“LBD”) separated from the DBD by a hinge region. As usedherein, the term “DNA binding domain” comprises a minimal polypeptidesequence of a DNA binding protein, up to the entire length of a DNAbinding protein, so long as the DNA binding domain functions toassociate with a particular response element. Members of the nuclearreceptor superfamily are also characterized by the presence of four orfive domains: A/B, C, D, E, and in some members F (see U.S. Pat. No.4,981,784 and Evans, Science 240:889-895 (1988)). The “A/B” domaincorresponds to the transactivation domain, “C” corresponds to the DNAbinding domain, “D” corresponds to the hinge region, and “E” correspondsto the ligand binding domain. Some members of the family may also haveanother transactivation domain on the carboxy-terminal side of the LBDcorresponding to “F”.

The DBD is characterized by the presence of two cysteine zinc fingersbetween which are two amino acid motifs, the P-box and the D-box, whichconfer specificity for ecdysone response elements. These domains may beeither native, modified, or chimeras of different domains ofheterologous receptor proteins. The EcR receptor, like a subset of thesteroid receptor family, also possesses less well-defined regionsresponsible for heterodimerization properties. Because the domains ofnuclear receptors are modular in nature, the LBD, DBD, andtransactivation domains may be interchanged.

Gene switch systems are known that incorporate components from theecdysone receptor complex. However, in these known systems, whenever EcRis used it is associated with native or modified DNA binding domains andtransactivation domains on the same molecule. USP or RXR are typicallyused as silent partners. Applicants have previously shown that when DNAbinding domains and transactivation domains are on the same molecule thebackground activity in the absence of ligand is high and that suchactivity is dramatically reduced when DNA binding domains andtransactivation domains are on different molecules, that is, on each oftwo partners of a heterodimeric or homodimeric complex (seePCT/US01/09050).

Gene Expression Cassettes of the Invention

The novel nuclear receptor-based inducible gene expression system of theinvention comprises at least one gene expression cassette that iscapable of being expressed in a host cell, wherein the gene expressioncassette comprises a polynucleotide that encodes a polypeptidecomprising a Group H nuclear receptor ligand binding domain comprising asubstitution mutation. Thus, Applicants' invention also provides novelgene expression cassettes for use in the gene expression system of theinvention.

In a specific embodiment, the gene expression cassette that is capableof being expressed in a host cell comprises a polynucleotide thatencodes a polypeptide selected from the group consisting of a) apolypeptide comprising a transactivation domain, a DNA-binding domain,and a Group H nuclear receptor ligand binding domain comprising asubstitution mutation; b) a polypeptide comprising a DNA-binding domainand a Group H nuclear receptor ligand binding domain comprising asubstitution mutation; and c) a polypeptide comprising a transactivationdomain and a Group H nuclear receptor ligand binding domain comprising asubstitution mutation.

In another specific embodiment, the present invention provides a geneexpression cassette that is capable of being expressed in a host cell,wherein the gene expression cassette comprises a polynucleotide thatencodes a hybrid polypeptide selected from the group consisting of a) ahybrid polypeptide comprising a transactivation domain, a DNA-bindingdomain, and a Group H nuclear receptor ligand binding domain comprisinga substitution mutation; b) a hybrid polypeptide comprising aDNA-binding domain and a Group H nuclear receptor ligand binding domaincomprising a substitution mutation; and c) a hybrid polypeptidecomprising a transactivation domain and a Group H nuclear receptorligand binding domain comprising a substitution mutation. A hybridpolypeptide according to the invention comprises at least twopolypeptide fragments, wherein each polypeptide fragment is from adifferent source, i.e., a different polypeptide, a different nuclearreceptor, a different species, etc. The hybrid polypeptide according tothe invention may comprise at least two polypeptide domains, whereineach polypeptide domain is from a different source.

In a specific embodiment, the Group H nuclear receptor ligand bindingdomain comprising a substitution mutation is from an ecdysone receptor,a ubiquitous receptor, an orphan receptor 1, a NER-1, a steroid hormonenuclear receptor 1, a retinoid X receptor interacting protein −15, aliver X receptor β, a steroid hormone receptor like protein, a liver Xreceptor, a liver X receptor α, a farnesoid X receptor, a receptorinteracting protein 14, and a farnesol receptor. In a preferredembodiment, the Group H nuclear receptor ligand binding domain is froman ecdysone receptor.

Thus, the present invention also provides a gene expression cassettecomprising a polynucleotide that encodes a polypeptide selected from thegroup consisting of a) a polypeptide comprising a transactivationdomain, a DNA-binding domain, and an ecdysone receptor ligand bindingdomain comprising a substitution mutation; b) a polypeptide comprising aDNA-binding domain and an ecdysone receptor ligand binding domaincomprising a substitution mutation; and c) a polypeptide comprising atransactivation domain and an ecdysone receptor ligand binding domaincomprising a substitution mutation. Preferably, the gene expressioncassette comprises a polynucleotide that encodes a hybrid polypeptideselected from the group consisting of a) a hybrid polypeptide comprisinga transactivation domain, a DNA-binding domain, and an ecdysone receptorligand binding domain comprising a substitution mutation; b) a hybridpolypeptide comprising a DNA-binding domain and an ecdysone receptorligand binding domain comprising a substitution mutation; and c) ahybrid polypeptide comprising a transactivation domain and an ecdysonereceptor ligand binding domain comprising a substitution mutation;wherein the encoded hybrid polypeptide comprises at least twopolypeptide fragments, wherein each polypeptide fragment is from adifferent source.

The ecdysone receptor (EcR) ligand binding domain (LBD) may be from aninvertebrate EcR, preferably selected from the class Arthropod EcR.Preferably the EcR is selected from the group consisting of aLepidopteran EcR, a Dipteran EcR, an Orthopteran EcR, a Homopteran EcRand a Hemipteran EcR. More preferably, the EcR ligand binding domain foruse in the present invention is from a spruce budworm Choristoneurafumiferana EcR (“CfEcR”), a beetle Tenebrio molitor EcR (“TmEcR”), aManduca sexta EcR (“MsEcR”), a Heliothies virescens EcR (“HvEcR”), amidge Chironomus tentans EcR (“CtEcR”), a silk moth Bombyx mori EcR(“BmEcR”), a squinting bush brown Bicyclus anynana EcR (“BanEcR”), abuckeye Junonia coenia EcR (“JcEcR”), a fruit fly Drosophilamelanogaster EcR (“DmEcR”), a mosquito Aedes aegypti EcR (“AaEcR”), ablowfly Lucilia capitata (“LcEcR”), a blowfly Lucilia cuprina EcR(“LucEcR”), a blowfly Calliphora vicinia EcR (“CvEcR”), a Mediterraneanfruit fly Ceratitis capitata EcR (“CcEcR”), a locust Locusta migratoriaEcR (“LmEcR”), an aphid Myzus persicae EcR (“MpEcR”), a fiddler crabCeluca pugilator EcR (“CpEcR”), an ixodid tick Amblyomma americanum EcR(“AmaEcR”), a whitefly Bamecia argentifoli EcR (“BaEcR”, SEQ ID NO: 112)or a leafhopper Nephotetix cincticeps EcR (“NcEcR”, SEQ ID NO: 113).More preferably, the LBD is from a CfEcR, a DmEcR, or an AmaEcR.

In a specific embodiment, the LBD is from a truncated EcR polypeptide.The EcR polypeptide truncation results in a deletion of at least 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, or 265 amino acids. Preferably, the EcRpolypeptide truncation results in a deletion of at least a partialpolypeptide domain. More preferably, the EcR polypeptide truncationresults in a deletion of at least an entire polypeptide domain. In aspecific embodiment, the EcR polypeptide truncation results in adeletion of at least an A/B-domain, a C-domain, a D-domain, an F-domain,an A/B/C-domains, an A/B/1/2-C-domains, an A/B/C/D-domains, anA/B/C/D/F-domains, an A/B/F-domains, an A/B/C/F-domains, a partial Edomain, or a partial F domain. A combination of several complete and/orpartial domain deletions may also be performed.

In a specific embodiment, the Group H nuclear receptor ligand bindingdomain is encoded by a polynucleotide comprising a codon mutation thatresults in a substitution of a) amino acid residue 20, 21, 48, 51, 52,55, 58, 59, 61, 62, 92, 93, 95, 96, 107, 109, 110, 120, 123, 125, 175,218, 219, 223, 230, 234, or 238 of SEQ ID NO: 1, b) amino acid residues95 and 110 of SEQ ID NO: 1, c) amino acid residues 218 and 219 of SEQ IDNO: 1, d) amino acid residues 107 and 175 of SEQ ID NO: 1, e) amino acidresidues 127 and 175 of SEQ ID NO: 1, f) amino acid residues 107 and 127of SEQ ID NO: 1, g) amino acid residues 107, 127 and 175 of SEQ ID NO:1, h) amino acid residues 52, 107 and 175 of SEQ ID NO: 1, i) amino acidresidues 96, 107, and 175 of SEQ ID NO: 1, j) amino acid residues 107,110, and 175 of SEQ ID NO: 1, k) amino acid residue 107, 121, 213, or217 of SEQ ID NO: 2, or l) amino acid residue 91 or 105 of SEQ ID NO: 3.In a preferred embodiment, the Group H nuclear receptor ligand bindingdomain is from an ecdysone receptor.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain is encoded by a polynucleotide comprising a codonmutation that results in a substitution of a) an alanine residue at aposition equivalent or analogous to amino acid residue 20, 21, 48, 51,55, 58, 59, 61, 62, 92, 93, 95, 109, 120, 125, 218, 219, 223, 230, 234,or 238 of SEQ ID NO: 1, b) an alanine, valine, isoleucine, or leucineresidue at a position equivalent or analogous to amino acid residue 52of SEQ ID NO: 1, c) an alanine, threonine, aspartic acid, or methionineresidue at a position equivalent or analogous to amino acid residue 96of SEQ ID NO: 1, d) a proline, serine, methionine, or leucine residue ata position equivalent or analogous to amino acid residue 110 of SEQ IDNO: 1, e) a phenylalanine residue at a position equivalent or analogousto amino acid residue 123 of SEQ ID NO: 1, f) an alanine residue at aposition equivalent or analogous to amino acid residue 95 of SEQ ID NO:1 and a proline residue at a position equivalent or analogous to aminoacid residue 110 of SEQ ID NO: 1, g) an alanine residue at a positionequivalent or analogous to amino acid residues 218 and 219 of SEQ ID NO:1, h) an isoleucine residue at a position equivalent or analogous toamino acid residue 107 of SEQ ID NO: 1, i) an glutamine residue at aposition equivalent or analogous to amino acid residues 175 of SEQ IDNO: 1, j) an isoleucine residue at a position equivalent or analogous toamino acid residue 107 of SEQ ID NO: 1 and a glutamine residue at aposition equivalent or analogous to amino acid residue 175 of SEQ ID NO:1, k) a glutamine residue at a position equivalent or analogous to aminoacid residues 127 and 175 of SEQ ID NO: 1, l) an isoleucine residue at aposition equivalent or analogous to amino acid residue 107 of SEQ ID NO:1 and a glutamine residue at a position equivalent or analogous to aminoacid residue 127 of SEQ ID NO: 1, m) an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamine residue at a position equivalent or analogous to amino acidresidues 127 and 175 of SEQ ID NO: 1, n) a valine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1 and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, o) analanine residue at a position equivalent or analogous to amino acidresidue 96 of SEQ ID NO: 1, an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamine residue at a position equivalent or analogous to amino acidresidue 175 of SEQ ID NO: 1, p) an alanine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1, and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, q) athreonine residue at a position equivalent or analogous to amino acidresidue 96 of SEQ ID NO: 1, an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1, and aglutamine residue at a position equivalent or analogous to amino acidresidue 175 of SEQ ID NO: 1, r) an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1, aproline residue at a position equivalent or analogous to amino acid 110of SEQ ID NO: 1, and a glutamine residue at a position equivalent oranalogous to amino acid 175 of SEQ ID NO: 1, s) a proline at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 2, t) anarginine or a leucine at a position equivalent or analogous to aminoacid residue 121 of SEQ ID NO: 2, u) an alanine at a position equivalentor analogous to amino acid residue 213 of SEQ ID NO: 2, v) an alanine ora serine at a position equivalent or analogous to amino acid residue 217of SEQ ID NO: 2, w) an alanine at a position equivalent or analogous toamino acid residue 91 of SEQ ID NO: 3, or x) a proline at a positionequivalent or analogous to amino acid residue 105 of SEQ ID NO: 3. In apreferred embodiment, the Group H nuclear receptor ligand binding domainis from an ecdysone receptor.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is an ecdysonereceptor ligand binding domain comprising a substitution mutationencoded by a polynucleotide comprising a codon mutation that results ina substitution mutation selected from the group consisting of a) E20A,Q21A, F48A, I51A, T52A, T52V, T521, T52L, T55A, T58A, V59A, L61A, I62A,M92A, M93A, R95A, V96A, V96T, V96D, V96M, V107I, F109A, A110P, A110S,A110M, A110L, Y120A, A123F, M125A, R175E, M218A, C219A, L223A, L230A,L234A, W238A, R95A/A110P, M218A/C219A, V107I/R175E, Y127E/R175E,V107I/Y127E, V107I/Y127E/R175E, T52V/V107I/R175E, V96A/V107I/R175E,T52A/V107I/R175E, V96T/V107I/R175E or V107I/A110P/R175E substitutionmutation of SEQ ID NO: 1, b) A107P, G121R, G121L, N213A, C217A, or C217Ssubstitution mutation of SEQ ID NO: 2, and c) G91A or A105P substitutionmutation of SEQ ID NO: 3.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is an ecdysonereceptor ligand binding domain polypeptide comprising a substitutionmutation encoded by a polynucleotide that hybridizes to a polynucleotidecomprising a codon mutation that results in a substitution mutationselected from the group consisting of a) T58A, A110P, A110L, A110S, orA110M of SEQ ID NO: 1, b) A107P of SEQ ID NO: 2, and c) A105P of SEQ IDNO: 3 under hybridization conditions comprising a hybridization step inless than 500 mM salt and at least 37 degrees Celsius, and a washingstep in 2×SSPE at least 63 degrees Celsius. In a preferred embodiment,the hybridization conditions comprise less than 200 mM salt and at least37 degrees Celsius for the hybridization step. In another preferredembodiment, the hybridization conditions comprise 2×SSPE and 63 degreesCelsius for both the hybridization and washing steps. In anotherpreferred embodiment, the ecdysone receptor ligand binding domain lackssteroid binding activity, such as 20-hydroxyecdysone binding activity,ponasterone A binding activity, or muristerone A binding activity.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprises a substitution mutation at a positionequivalent or analogous to a) amino acid residue 20, 21, 48, 51, 52, 55,58, 59, 61, 62, 92, 93, 95, 96, 107, 109, 110, 120, 123, 125, 175, 218,219, 223, 230, 234, or 238 of SEQ ID NO: 1, b) amino acid residues 95and 110 of SEQ ID NO: 1, c) amino acid residues 218 and 219 of SEQ IDNO: 1, d) amino acid residues 107 and 175 of SEQ ID NO: 1, e) amino acidresidues 127 and 175 of SEQ ID NO: 1, f) amino acid residues 107 and 127of SEQ ID NO: 1, g) amino acid residues 107, 127 and 175 of SEQ ID NO:1, h) amino acid residues 52, 107 and 175 of SEQ ID NO: 1, i) amino acidresidues 96, 107 and 175 of SEQ ID NO: 1, j) amino acid residues 107,110, and 175 of SEQ ID NO: 1, k) amino acid residue 107, 121, 213, or217 of SEQ ID NO: 2, or l) amino acid residue 91 or 105 of SEQ ID NO: 3.In a preferred embodiment, the Group H nuclear receptor ligand bindingdomain is from an ecdysone receptor.

Preferably, the Group H nuclear receptor ligand binding domain comprisesa substitution of a) an alanine residue at a position equivalent oranalogous to amino acid residue 20, 21, 48, 51, 55, 58, 59, 61, 62, 92,93, 95, 109, 120, 125, 218, 219, 223, 230, 234, or 238 of SEQ ID NO: 1,b) an alanine, valine, isoleucine, or leucine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, c) analanine, threonine, aspartic acid, or methionine residue at a positionequivalent or analogous to amino acid residue 96 of SEQ ID NO: 1, d) aproline, serine, methionine, or leucine residue at a position equivalentor analogous to amino acid residue 110 of SEQ ID NO: 1, e) aphenylalanine residue at a position equivalent or analogous to aminoacid residue 123 of SEQ ID NO: 1, f) an alanine residue at a positionequivalent or analogous to amino acid residue 95 of SEQ ID NO: 1 and aproline residue at a position equivalent or analogous to amino acidresidue 110 of SEQ ID NO: 1, g) an alanine residue at a positionequivalent or analogous to amino acid residues 218 and 219 of SEQ ID NO:1, h) an isoleucine residue at a position equivalent or analogous toamino acid residue 107 of SEQ ID NO: 1, i) a glutamine residue at aposition equivalent or analogous to amino acid residues 175, j) anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1 and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, k) aglutamine residue at a position equivalent or analogous to amino acidresidues 127 and 175 of SEQ ID NO: 1, l) an isoleucine residue at aposition equivalent or analogous to amino acid residue 107 of SEQ ID NO:1 and a glutamine residue at a position equivalent or analogous to aminoacid residue 127 of SEQ ID NO: 1, m) an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamine residue at a position equivalent or analogous to amino acidresidues 127 and 175 of SEQ ID NO: 1, n) a valine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1 and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, o) analanine residue at a position equivalent or analogous to amino acidresidue 96 of SEQ ID NO: 1, an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamine residue at a position equivalent or analogous to amino acidresidue 175 of SEQ ID NO: 1, p) an alanine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1, and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, q) athreonine residue at a position equivalent or analogous to amino acidresidue 96 of SEQ ID NO: 1, an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1, and aglutamine residue at a position equivalent or analogous to amino acidresidue 175 of SEQ ID NO: 1, r) an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1, aproline residue at a position equivalent or analogous to amino acid 110of SEQ ID NO: 1, and a glutamine residue at a position equivalent oranalogous to amino acid 175 of SEQ ID NO: 1, s) a proline at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 2, t) anarginine or a leucine at a position equivalent or analogous to aminoacid residue 121 of SEQ ID NO: 2, u) an alanine at a position equivalentor analogous to amino acid residue 213 of SEQ ID NO: 2, v) an alanine ora serine at a position equivalent or analogous to amino acid residue 217of SEQ ID NO: 2, w) an alanine at a position equivalent or analogous toamino acid residue 91 of SEQ ID NO: 3, or x) a proline at a positionequivalent or analogous to amino acid residue 105 of SEQ ID NO: 3. In apreferred embodiment, the Group H nuclear receptor ligand binding domainis from an ecdysone receptor.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is an ecdysonereceptor ligand binding domain polypeptide comprising a substitutionmutation, wherein the substitution mutation is selected from the groupconsisting of a) E20A, Q21A, F48A, I51A, T52A, T52V, T52I, T52L, T55A,T58A, V59A, L61A, I62A, M92A, M93A, R95A, V96A, V96T, V96D, V96M, V107I,F109A, A110P, A110S, A110M, A110L, Y120A, A123F, M125A, R175E, M218A,C219A, L223A, L230A, L234A, W238A, R95A/A110P, M218A/C219A, V107I/R175E,Y127E/R175E, V107I/Y127E, V107I/Y127E/R175E, T52V/V107I/R175E,V96A/V107I/R175E, T52A/V107I/R175E, V96T/V107I/R175E, orV107I/A110P/R175E substitution mutation of SEQ ID NO: 1, b) A107P,G121R, G121L, N213A, C217A, or C217S substitution mutation of SEQ ID NO:2, and c) G91A or A105P substitution mutation of SEQ ID NO: 3.

The DNA binding domain can be any DNA binding domain with a knownresponse element, including synthetic and chimeric DNA binding domains,or analogs, combinations, or modifications thereof. Preferably, the DBDis a GAL4 DBD, a LexA DBD, a transcription factor DBD, a Group H nuclearreceptor member DBD, a steroid/thyroid hormone nuclear receptorsuperfamily member DBD, or a bacterial LacZ DBD. More preferably, theDBD is an EcR DBD [SEQ ID NO: 4 (polynucleotide) or SEQ ID NO: 5(polypeptide)], a GAL4 DBD [SEQ ID NO: 6 (polynucleotide) or SEQ ID NO:7 (polypeptide)], or a LexA DBD [(SEQ ID NO: 8 (polynucleotide) or SEQID NO: 9 (polypeptide)].

The transactivation domain (abbreviated “AD” or “TA”) may be any Group Hnuclear receptor member AD, steroid/thyroid hormone nuclear receptor AD,synthetic or chimeric AD, polyglutamine AD, basic or acidic amino acidAD, a VP16 AD, a GAL4 AD, an NF-κB AD, a BP64 AD, a B42 acidicactivation domain (B42AD), a p65 transactivation domain (p65AD), or ananalog, combination, or modification thereof. In a specific embodiment,the AD is a synthetic or chimeric AD, or is obtained from an EcR, aglucocorticoid receptor, VP16, GAL4, NF-kB, or B42 acidic activationdomain AD. Preferably, the AD is an EcR AD [SEQ ID NO: 10(polynucleotide) or SEQ ID NO: 11 (polypeptide)], a VP16 AD [SEQ ID NO:12 (polynucleotide) or SEQ ID NO: 13 (polypeptide)], a B42 AD [SEQ IDNO: 14 (polynucleotide) or SEQ ID NO: 15 (polypeptide)], or a p65 AD[SEQ ID NO: 16 (polynucleotide) or SEQ ID NO: 17 (polypeptide)].

In a specific embodiment, the gene expression cassette encodes a hybridpolypeptide comprising either a) a DNA-binding domain encoded by apolynucleotide comprising a nucleic acid sequence of SEQ ID NO: 4, SEQID NO: 6, or SEQ ID NO: 8, or b) a transactivation domain encoded by apolynucleotide comprising a nucleic acid sequence of SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; and a Group H nuclearreceptor ligand binding domain comprising a substitution mutationencoded by a polynucleotide according to the invention. Preferably, theGroup H nuclear receptor ligand binding domain comprising a substitutionmutation is an ecdysone receptor ligand binding domain comprising asubstitution mutation encoded by a polynucleotide according to theinvention.

In another specific embodiment, the gene expression cassette encodes ahybrid polypeptide comprising either a) a DNA-binding domain comprisingan amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,or b) a transactivation domain comprising an amino acid sequence of SEQID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17; and a Group Hnuclear receptor ligand binding domain comprising a substitutionmutation according to the invention. Preferably, the Group H nuclearreceptor ligand binding domain comprising a substitution mutation is anecdysone receptor ligand binding domain comprising a substitutionmutation according to the invention.

The present invention also provides a gene expression cassettecomprising: i) a response element comprising a domain recognized by apolypeptide comprising a DNA binding domain; ii) a promoter that isactivated by a polypeptide comprising a transactivation domain; and iii)a gene whose expression is to be modulated.

The response element (“RE”) may be any response element with a known DNAbinding domain, or an analog, combination, or modification thereof. Asingle RE may be employed or multiple REs, either multiple copies of thesame RE or two or more different REs, may be used in the presentinvention. In a specific embodiment, the RE is an RE from GAL4(“GAL4RE”), LexA, a Group H nuclear receptor RE, a steroid/thyroidhormone nuclear receptor RE, or a synthetic RE that recognizes asynthetic DNA binding domain. Preferably, the RE is an ecdysone responseelement (EcRE) comprising a polynucleotide sequence of SEQ ID NO: 18, aGAL4RE comprising a polynucleotide sequence of SEQ ID NO: 19, or a LexARE (operon, “op”) comprising a polynucleotide sequence of SEQ ID NO: 20(“2XLexAopRE”).

A steroid/thyroid hormone nuclear receptor DNA binding domain,activation domain or response element according to the invention may beobtained from a steroid/thyroid hormone nuclear receptor selected fromthe group consisting of thyroid hormone receptor α (TRα), thyroidreceptor 1 (c-erbA-1), thyroid hormone receptor β (TRβ), retinoic acidreceptor α (RARα), retinoic acid receptor β (RARβ, HAP), retinoic acidreceptor γ (RARγ), retinoic acid receptor gamma-like (RARD), peroxisomeproliferator-activated receptor α (PPARα), peroxisomeproliferator-activated receptor β (PPARβ), peroxisomeproliferator-activated receptor δ (PPARδ, NUC-1), peroxisomeproliferator-activator related receptor (FFAR), peroxisomeproliferator-activated receptor γ (PPARγ), orphan receptor encoded bynon-encoding strand of thyroid hormone receptor α (REVERBα), v-erb Arelated receptor (EAR-1), v-erb related receptor (EAR-1A), γ), orphanreceptor encoded by non-encoding strand of thyroid hormone receptor β(REVERBβ), v-erb related receptor (EAR-1β), orphan nuclear receptor BD73(BD73), rev-erbA-related receptor (RVR), zinc finger protein 126 (HZF2),ecdysone-inducible protein E75 (E75), ecdysone-inducible protein E78(E78), Drosophila receptor 78 (DR-78), retinoid-related orphan receptorα (RORα), retinoid Z receptor α (RZRα), retinoid related orphan receptorβ (RORβ), retinoid Z receptor β (RZRβ), retinoid-related orphan receptorγ (RORγ), retinoid Z receptor γ (RZRγ), retinoid-related orphan receptor(TOR), hormone receptor 3 (HR-3), Drosophila hormone receptor 3 (DHR-3),Manduca hormone receptor (MHR-3), Galleria hormone receptor 3 (GHR-3),C. elegans nuclear receptor 3 (CNR-3), Choristoneura hormone receptor 3(CHR-3), C. elegans nuclear receptor 14 (CNR-14), ecdysone receptor(ECR), ubiquitous receptor (UR), orphan nuclear receptor (OR-1), NER-1,receptor-interacting protein 15 (RIP-15), liver X receptor β (LXRβ),steroid hormone receptor like protein (RLD-1), liver X receptor (LXR),liver X receptor α (LXRα), farnesoid X receptor (FXR),receptor-interacting protein 14 (RIP-14), HRR-1, vitamin D receptor(VDR), orphan nuclear receptor (ONR-1), pregnane X receptor (PXR),steroid and xenobiotic receptor (SXR), benzoate X receptor (BXR),nuclear receptor (MB-67), constitutive androstane receptor 1 (CAR-1),constitutive androstane receptor α (CARα), constitutive androstanereceptor 2 (CAR-2), constitutive androstane receptor β (CARβ),Drosophila hormone receptor 96 (DHR-96), nuclear hormone receptor 1(NHR-1), hepatocyte nuclear factor 4 (HNF-4), hepatocyte nuclear factor4G (HNF-4G), hepatocyte nuclear factor 4B (HNF-4B), hepatocyte nuclearfactor 4D (HNF-4D, DHNF-4), retinoid X receptor α (RXRα), retinoid Xreceptor β (RXRβ), H-2 region II binding protein (H-2RIIBP), nuclearreceptor co-regulator-1 (RCoR-1), retinoid X receptor γ (RXRγ),Ultraspiracle (USP), 2C1 nuclear receptor, chorion factor 1 (CF-1),testicular receptor 2 (TR-2), testicular receptor 2-11 (TR2-11),testicular receptor 4 (TR4), TAK-1, Drosophila hormone receptor (DHR78),Tailless (TLL), tailless homolog (TLX), XTLL, chicken ovalbumin upstreampromoter transcription factor I (COUP-TFI), chicken ovalbumin upstreampromoter transcription factor A (COUP-TFA), EAR-3, SVP-44, chickenovalbumin upstream promoter transcription factor II (COUP-TFII), chickenovalbumin upstream promoter transcription factor B (COUP-TFB), ARP-1,SVP-40, SVP, chicken ovalbumin upstream promoter transcription factorIII (COUP-TFIII), chicken ovalbumin upstream promoter transcriptionfactor G (COUP-TFG), SVP-46, EAR-2, estrogen receptor α (ERα), estrogenreceptor β (ERβ), estrogen related receptor 1 (ERR1), estrogen relatedreceptor α (ERRα), estrogen related receptor 2 (ERR2), estrogen relatedreceptor β (ERRβ), glucocorticoid receptor (GR), mineralocorticoidreceptor (MR), progesterone receptor (PR), androgen receptor (AR), nervegrowth factor induced gene B (NGFI-B), nuclear receptor similar toNur-77 (TRS), N10, Orphan receptor (NUR-77), Human early response gene(NAK-1), Nun related factor 1 (NURR-1), a human immediate-early responsegene (NOT), regenerating liver nuclear receptor 1 (RNR-1), hematopoieticzinc finger 3 (HZF-3), Nur rekated protein-1 (TINOR), Nuclear orphanreceptor 1 (NOR-1), NOR1 related receptor (MINOR), Drosophila hormonereceptor 38 (DHR-38), C. elegans nuclear receptor 8 (CNR-8), C48D5,steroidogenic factor 1 (SF1), endozepine-like peptide (ELP), fushitarazu factor 1 (FTZ-F1), adrenal 4 binding protein (AD4BP), liverreceptor homolog (LRH-1), Ftz-F1-related orphan receptor A (xFFrA),Ftz-F1-related orphan receptor B (xFFrB), nuclear receptor related toLRH-1 (FFLR), nuclear receptor related to LRH-1 (PHR), fetoproteintranscriptin factor (FTF), germ cell nuclear factor (GCNFM), retinoidreceptor-related testis-associated receptor (RTR), knirps (KNI), knirpsrelated (KNRL), Embryonic gonad (EGON), Drosophila gene for liganddependent nuclear receptor (EAGLE), nuclear receptor similar totrithorax (ODR7), Trithorax, dosage sensitive sex reversal adrenalhypoplasia congenita critical region chromosome X gene (DAX-1), adrenalhypoplasia congenita and hypogonadotropic hypogonadism (ARCH), and shortheterodimer partner (SHP).

For purposes of this invention, nuclear receptors and Group H nuclearreceptors also include synthetic and chimeric nuclear receptors andGroup H nuclear receptors and their homologs.

Genes of interest for use in Applicants' gene expression cassettes maybe endogenous genes or heterologous genes. Nucleic acid or amino acidsequence information for a desired gene or protein can be located in oneof many public access databases, for example, GENBANK, EMBL, Swiss-Prot,and PIR, or in many biology related journal publications. Thus, thoseskilled in the art have access to nucleic acid sequence information forvirtually all known genes. Such information can then be used toconstruct the desired constructs for the insertion of the gene ofinterest within the gene expression cassettes used in Applicants'methods described herein.

Examples of genes of interest for use in Applicants' gene expressioncassettes include, but are not limited to: genes encodingtherapeutically desirable polypeptides or products that may be used totreat a condition, a disease, a disorder, a dysfunction, a geneticdefect, such as monoclonal antibodies, enzymes, proteases, cytokines,interferons, insulin, erthropoietin, clotting factors, other bloodfactors or components, viral vectors for gene therapy, virus forvaccines, targets for drug discovery, functional genomics, andproteomics analyses and applications, and the like.

Polynucleotides of the Invention

The novel nuclear receptor-based inducible gene expression system of theinvention comprises at least one gene expression cassette comprising apolynucleotide that encodes a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation. These gene expressioncassettes, the polynucleotides they comprise, and the polypeptides theyencode are useful as components of a nuclear receptor-based geneexpression system to modulate the expression of a gene within a hostcell.

Thus, the present invention provides an isolated polynucleotide thatencodes a Group H nuclear receptor ligand binding domain comprising asubstitution mutation.

In a specific embodiment, the Group H nuclear receptor ligand bindingdomain is encoded by a polynucleotide comprising a codon mutation thatresults in a substitution of an amino acid residue at a positionequivalent or analogous to a) amino acid residue 20, 21, 48, 51, 52, 55,58, 59, 61, 62, 92, 93, 95, 96, 107, 109, 110, 120, 123, 125, 175, 218,219, 223, 230, 234, or 238 of SEQ ID NO: 1, b) amino acid residues 95and 110 of SEQ ID NO: 1, c) amino acid residues 218 and 219 of SEQ IDNO: 1, d) amino acid residues 107 and 175 of SEQ ID NO: 1, e) amino acidresidues 127 and 175 of SEQ ID NO: 1, f) amino acid residues 107 and 127of SEQ ID NO: 1, g) amino acid residues 107, 127 and 175 of SEQ ID NO:1, h) amino acid residues 52, 107 and 175 of SEQ ID NO: 1, i) amino acidresidues 96, 107 and 175 of SEQ ID NO: 1, j) amino acid residues 107,110, and 175 of SEQ ID NO: 1, k) amino acid residue 107, 121, 213, or217 of SEQ ID NO: 2, or l) amino acid residue 91 or 105 of SEQ ID NO: 3.In a preferred embodiment, the Group H nuclear receptor ligand bindingdomain is from an ecdysone receptor.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain is encoded by a polynucleotide comprising a codonmutation that results in a substitution of a) an alanine residue at aposition equivalent or analogous to amino acid residue 20, 21, 48, 51,55, 58, 59, 61, 62, 92, 93, 95, 109, 120, 125, 218, 219, 223, 230, 234,or 238 of SEQ ID NO: 1, b) an alanine, valine, isoleucine, or leucineresidue at a position equivalent or analogous to amino acid residue 52of SEQ ID NO: 1, c) an alanine, threonine, aspartic acid, or methionineresidue at a position equivalent or analogous to amino acid residue 96of SEQ ID NO: 1, d) a proline, serine, methionine, or leucine residue ata position equivalent or analogous to amino acid residue 110 of SEQ IDNO: 1, e) a phenylalanine residue at a position equivalent or analogousto amino acid residue 123 of SEQ ID NO: 1, f) an alanine residue at aposition equivalent or analogous to amino acid residue 95 of SEQ ID NO:1 and a proline residue at a position equivalent or analogous to aminoacid residue 110 of SEQ ID NO: 1, g) an alanine residue at a positionequivalent or analogous to amino acid residues 218 and 219 of SEQ ID NO:1, h) an isoleucine residue at a position equivalent or analogous toamino acid residue 107 of SEQ ID NO: 1, i) a glutamine residue at aposition equivalent or analogous to amino acid residues 175, j) anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1 and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, k) aglutamine residue at a position equivalent or analogous to amino acidresidues 127 and 175 of SEQ ID NO: 1, l) an isoleucine residue at aposition equivalent or analogous to amino acid residue 107 of SEQ ID NO:1 and a glutamine residue at a position equivalent or analogous to aminoacid residue 127 of SEQ ID NO: 1, m) an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamine residue at a position equivalent or analogous to amino acidresidues 127 and 175 of SEQ ID NO: 1, n) a valine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1 and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, o) analanine residue at a position equivalent or analogous to amino acidresidue 96 of SEQ ID NO: 1, an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamine residue at a position equivalent or analogous to amino acidresidue 175 of SEQ ID NO: 1, p) an alanine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1, and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, q) athreonine residue at a position equivalent or analogous to amino acidresidue 96 of SEQ ID NO: 1, an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1, and aglutamine residue at a position equivalent or analogous to amino acidresidue 175 of SEQ ID NO: 1, r) an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1, aproline residue at a position equivalent or analogous to amino acid 110of SEQ ID NO: 1, and a glutamine residue at a position equivalent oranalogous to amino acid 175 of SEQ ID NO: 1, s) a proline at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 2, t) anarginine or a leucine at a position equivalent or analogous to aminoacid residue 121 of SEQ ID NO: 2, u) an alanine at a position equivalentor analogous to amino acid residue 213 of SEQ ID NO: 2, v) an alanine ora serine at a position equivalent or analogous to amino acid residue 217of SEQ ID NO: 2, w) an alanine at a position equivalent or analogous toamino acid residue 91 of SEQ ID NO: 3, or x) a proline at a positionequivalent or analogous to amino acid residue 105 of SEQ ID NO: 3. In apreferred embodiment, the Group H nuclear receptor ligand binding domainis from an ecdysone receptor.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is an ecdysonereceptor ligand binding domain comprising a substitution mutationencoded by a polynucleotide comprising a codon mutation that results ina substitution mutation selected from the group consisting of a) E20A,Q21A, F48A, I51A, T52A, T52V, T52I, T52L, T55A, T58A, V59A, L61A, I62A,M92A, M93A, R95A, V96A, V96T, V96D, V96M, V107I, F109A, A110P, A110S,A110M, A110L, Y120A, A123F, M125A, R175E, M218A, C219A, L223A, L230A,L234A, W238A, R95A/A110P, M218A/C219A, V107I/R175E, Y127E/R175E,V107I/Y127E, V107I/Y127E/R175E, T52V/V107I/R175E, V96A/V107I/R175E,T52A/V107I/R175E, V96T/V107I/R175E or V107I/A110P/R175E substitutionmutation of SEQ ID NO: 1, b) A107P, G121R, G121L, N213A, C217A, or C217Ssubstitution mutation of SEQ ID NO: 2, and c) G91A or A105P substitutionmutation of SEQ ID NO: 3.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is an ecdysonereceptor ligand binding domain comprising a substitution mutationencoded by a polynucleotide that hybridizes to a polynucleotidecomprising a codon mutation that results in a substitution mutationselected from the group consisting of a) T58A, A110P, A110L, A110S, orA110M of SEQ ID NO: 1, b) A107P of SEQ ID NO: 2, and c) A105P of SEQ IDNO: 3 under hybridization conditions comprising a hybridization step inless than 500 mM salt and at least 37 degrees Celsius, and a washingstep in 2×SSPE at least 63 degrees Celsius. In a preferred embodiment,the hybridization conditions comprise less than 200 mM salt and at least37 degrees Celsius for the hybridization step. In another preferredembodiment, the hybridization conditions comprise 2×SSPE and 63 degreesCelsius for both the hybridization and washing steps. In anotherpreferred embodiment, the ecdysone receptor ligand binding domain lacksbinding activity to a steroid such as 20-hydroxyecdysone, ponasterone A,or muristerone A.

The present invention also provides an isolated polynucleotide thatencodes a polypeptide selected from the group consisting of a) apolypeptide comprising a transactivation domain, a DNA-binding domain,and a Group H nuclear receptor ligand binding domain comprising asubstitution mutation according to the invention; b) a polypeptidecomprising a DNA-binding domain and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation according to theinvention; and c) a polypeptide comprising a transactivation domain anda Group H nuclear receptor ligand binding domain comprising asubstitution mutation according to the invention.

In a specific embodiment, the isolated polynucleotide encodes a hybridpolypeptide selected from the group consisting of a) a hybridpolypeptide comprising a transactivation domain, a DNA-binding domain,and a Group H nuclear receptor ligand binding domain comprising asubstitution mutation according to the invention; b) a hybridpolypeptide comprising a DNA-binding domain and a Group H nuclearreceptor ligand binding domain comprising a substitution mutationaccording to the invention; and c) a hybrid polypeptide comprising atransactivation domain and a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation according to the invention.

The present invention also relates to an isolated polynucleotideencoding a Group H nuclear receptor ligand binding domain comprising asubstitution mutation, wherein the substitution mutation affects ligandbinding activity or ligand sensitivity of the Group H nuclear receptorligand binding domain.

In particular, the present invention relates to an isolatedpolynucleotide encoding a Group H nuclear receptor ligand binding domaincomprising a substitution mutation, wherein the substitution mutationreduces ligand binding activity or ligand sensitivity of the Group Hnuclear receptor ligand binding domain.

In a specific embodiment, the present invention relates to an isolatedpolynucleotide encoding a Group H nuclear receptor ligand binding domaincomprising a substitution mutation, wherein the substitution mutationreduces steroid binding activity or steroid sensitivity of the Group Hnuclear receptor ligand binding domain. Preferably, the isolatedpolynucleotide comprises a codon mutation that results in a substitutionof an amino acid residue at a position equivalent or analogous to a)amino acid residue 20, 21, 48, 51, 52, 55, 58, 59, 62, 92, 93, 95, 109,110, 120, 123, 125, 218, 219, 223, 230, 234, or 238 of SEQ ID NO: 1, b)amino acid residues 95 and 110 of SEQ ID NO: 1, c) amino acid residues218 and 219 of SEQ ID NO: 1, d) amino acid residue 107, 121, 213, or 217of SEQ ID NO: 2, or e) amino acid residue 105 of SEQ ID NO: 3. Morepreferably, the isolated polynucleotide comprises a codon mutation thatresults in a substitution of a) an alanine residue at a positionequivalent or analogous to amino acid residue 20, 21, 48, 51, 52, 55,58, 59, 62, 92, 93, 95, 109, 120, 125, 218, 219, 223, 230, 234, or 238of SEQ ID NO: 1, b) a proline residue at a position equivalent oranalogous to amino acid residue 110 of SEQ ID NO: 1, c) a phenylalanineresidue at a position equivalent or analogous to amino acid residue 123of SEQ ID NO: 1, d) an alanine residue at a position equivalent oranalogous to amino acid residue 95 of SEQ ID NO: 1 and a proline residueat a position equivalent or analogous to amino acid residue 110 of SEQID NO: 1, e) an alanine residue at a position equivalent or analogous toamino acid residues 218 and 219 of SEQ ID NO: 1, f) a proline residue ata position equivalent or analogous to amino acid residue 107 of SEQ IDNO: 2, g) an arginine or leucine residue at a position equivalent oranalogous to amino acid residue 121 of SEQ ID NO: 2, h) an alanineresidue at a position equivalent or analogous to amino acid residue 213of SEQ ID NO: 2, i) an alanine or a serine residue at a positionequivalent or analogous to amino acid residue 217 of SEQ ID NO: 2, or j)a proline residue at a position equivalent or analogous to amino acidresidue 105 of SEQ ID NO: 3. Even more preferably, the isolatedpolynucleotide comprises a codon mutation that results in a substitutionmutation of a) E20A, Q21A, F48A, I51A, T52A, T55A, T58A, V59A, I62A,M92A, M93A, R95A, F109A, A110P, Y120A, A123F, M125A, M218A, C219A,L223A, L230A, L234A, W238A, R95A/A110P, or M218A/C219A of SEQ ID NO: 1,b) A107P, G121R, G121L, N213A, C217A, or C217S of SEQ ID NO: 2, or c)A105P of SEQ ID NO: 3.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation eliminates steroid binding activity or steroidsensitivity of the Group H ligand binding domain. Preferably, theisolated polynucleotide comprises a codon mutation that results in asubstitution of an amino acid residue at a position equivalent oranalogous to a) amino acid residue 58 or 110 of SEQ ID NO: 1, b) aminoacid residues 107, 110 and 175 of SEQ ID NO: 1, c) amino acid residue107, 121, 213, or 217 of SEQ ID NO: 2, or d) amino acid residue 105 ofSEQ ID NO: 3. More preferably, the isolated polynucleotide comprises acodon mutation that results in a substitution of a) an alanine at aposition equivalent or analogous to amino acid residue 58 of SEQ ID NO:1, b) a proline, leucine, serine, or methionine residue at a positionequivalent or analogous to amino acid residue 110 of SEQ ID NO: 1, c) anisoleucine at a position equivalent or analogous to amino acid residue107 of SEQ ID NO: 1, a proline at a position equivalent or analogous toamino acid residue 110 of SEQ ID NO: 1, and a glutamine at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, d) aproline at a position equivalent or analogous to amino acid residue 107of SEQ ID NO: 2, e) an arginine or a leucine at a position equivalent oranalogous to amino acid residue 121 of SEQ ID NO: 2, f) an alanine at aposition equivalent or analogous to amino acid residue 213 of SEQ ID NO:2, g) an alanine or a serine at a position equivalent or analogous toamino acid residue 217 of SEQ ID NO: 2, or h) a proline at a positionequivalent or analogous to amino acid residue 105 of SEQ ID NO: 3. Evenmore preferably, the isolated polynucleotide comprises a codon mutationthat results in a substitution mutation selected from the groupconsisting of a) T58A, A110P, A110L, A110S, A110M, or V107I/A110P/R175Esubstitution mutation of SEQ ID NO: 1, b) A107P, G121R, G121L, N213A,C217A, or C217S substitution mutation of SEQ ID NO: 2, and c) A105Psubstitution mutation of SEQ ID NO: 3.

The present invention also relates to an isolated polynucleotideencoding a polypeptide comprising an ecdysone receptor ligand bindingdomain comprising a substitution mutation, wherein the ecdysone receptorligand binding domain lacks steroid binding activity. Preferably, theecdysone receptor ligand binding domain comprises a codon mutation thatresults in a substitution mutation at an equivalent or analogous aminoacid residue to a) amino acid residue 58 or 110 of SEQ ID NO: 1, b)amino acid residues 107, 110 and 175 of SEQ ID NO: 1, b) amino acidresidue 107, 121, 213, or 217 of SEQ ID NO: 2, or d) amino acid residue105 of SEQ ID NO: 3. More preferably, the ecdysone receptor ligandbinding domain comprises a codon mutation that results in a substitutionof a) an alanine at a position equivalent or analogous to amino acidresidue 58 of SEQ ID NO: 1, b) a proline, leucine, serine, or methionineresidue at a position equivalent or analogous to amino acid residue 110of SEQ ID NO: 1, c) an isoleucine at a position equivalent or analogousto amino acid residue 107 of SEQ ID NO: 1, a proline at a positionequivalent or analogous to amino acid residue 110 of SEQ ID NO: 1, aglutamine at a position equivalent or analogous to amino acid residue175 of SEQ ID NO: 1, d) a proline at a position equivalent or analogousto amino acid residue 107 of SEQ ID NO: 2, e) an arginine or a leucineat a position equivalent or analogous to amino acid residue 121 of SEQID NO: 2, f) an alanine at a position equivalent or analogous to aminoacid residue 213 of SEQ ID NO: 2, g) an alanine or a serine at aposition equivalent or analogous to amino acid residue 217 of SEQ ID NO:2, or h) a proline at a position equivalent or analogous to amino acidresidue 105 of SEQ ID NO: 3. Even more preferably, the isolated theecdysone receptor ligand binding domain comprises a codon mutation thatresults in a substitution mutation selected from the group consisting ofa) T58A, A110P, A110L, A110S, A110M, or V107I/A110P/R175E substitutionmutation of SEQ ID NO: 1, b) A107P, G121R, G121L, N213A, C217A, or C217Ssubstitution mutation of SEQ ID NO: 2, and c) A105P substitutionmutation of SEQ ID NO: 3. In a specific embodiment, the ecdysonereceptor ligand binding domain lacks steroid binding activity selectedfrom the group consisting of ecdysone binding activity,20-hydroxyecdysone binding activity, ponasterone A binding activity, andmuristerone A binding activity.

In another specific embodiment, the isolated polynucleotide encoding anecdysone receptor ligand binding domain comprising a substitutionmutation, wherein the ecdysone receptor ligand binding domain lackssteroid binding activity, hybridizes to a polynucleotide comprising acodon mutation that results in a substitution mutation selected from thegroup consisting of a) T58A, A110P, A110L, A110S, A110M, orV107I/A110P/R175E substitution mutation of SEQ ID NO: 1, b) A107P,G121R, G121L, N213A, C217A, or C217S substitution mutation of SEQ ID NO:2, and c) A105P substitution mutation of SEQ ID NO: 3 underhybridization conditions comprising a hybridization step in less than500 mM salt and at least 37 degrees Celsius, and a washing step in2×SSPE at at least 63 degrees Celsius. In a preferred embodiment, thehybridization conditions comprise less than 200 mM salt and at least 37degrees Celsius for the hybridization step. In another preferredembodiment, the hybridization conditions comprise 2×SSPE and 63 degreesCelsius for both the hybridization and washing steps. In anotherpreferred embodiment, the ecdysone receptor ligand binding domain lackssteroid binding activity selected from the group consisting of ecdysonebinding activity, 20-hydroxyecdysone binding activity, ponasterone Abinding activity, and muristerone A binding activity.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation reduces non-steroid binding activity ornon-steroid sensitivity of the Group H nuclear receptor ligand bindingdomain. Preferably, the isolated polynucleotide comprises a codonmutation that results in a substitution of an amino acid residue at aposition equivalent or analogous to amino acid residue a) 21, 48, 51,52, 59, 62, 93, 95, 96, 109, 120, 123, 125, 218, 219, 223, 230, 234, or238 of SEQ ID NO: 1, b) 121, 213, or 217 of SEQ ID NO: 2, or c) 105 ofSEQ ID NO: 3. More preferably, the isolated polynucleotide comprises acodon mutation that results in a substitution of a) an alanine residueat a position equivalent or analogous to amino acid residue 21, 48, 51,59, 62, 93, 95, 96, 109, 120, 125, 218, 219, 223, 230, 234, or 238 ofSEQ ID NO: 1, b) a leucine residue at at a position equivalent oranalogous to amino acid residue 52 of SEQ ID NO: 1, c) a threonineresidue at at a position equivalent or analogous to amino acid residue96 of SEQ ID NO: 1, d) a phenylalanine residue at at a positionequivalent or analogous to amino acid residue 123 of SEQ ID NO: 1, e) analanine residue at at a position equivalent or analogous to amino acidresidue 95 of SEQ ID NO: 1 and a proline residue at a positionequivalent or analogous to amino acid residue 110 of SEQ ID NO: 1, f) analanine residue at at a position equivalent or analogous to amino acidresidues 218 and 219 of SEQ ID NO: 1, g) an arginine or a leucineresidue at a position equivalent or analogous to amino acid residue 121of SEQ ID NO: 2, h) an alanine residue at a position equivalent oranalogous to amino acid residue 213 of SEQ ID NO: 2, i) an alanine or aserine residue at a position equivalent or analogous to amino acidresidue 217 of SEQ ID NO: 2, or j) a proline residue at a positionequivalent or analogous to amino acid residue 105 of SEQ ID NO: 3. Evenmore preferably, the isolated polynucleotide comprises a codon mutationthat results in a substitution mutation of a) Q21A, F48A, I51A, T52L,V59A, I62A, M93A, R95A, V96A, V96T, F109A, Y120A, A123F, M125A, M218A,C219A, L223A, L230A, L234A, W238A, R95A/A110P, or M218/C219A of SEQ IDNO: 1, b) G121R, G121L, N213A, C217A, or C217S of SEQ ID NO: 2, or c)A105P of SEQ ID NO: 3.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor polypeptideligand binding domain comprising a substitution mutation, wherein thesubstitution mutation eliminates non-steroid binding activity ornon-steroid sensitivity of the Group H ligand binding domain.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor polypeptideligand binding domain comprising a substitution mutation, wherein thesubstitution mutation reduces both steroid binding activity or steroidsensitivity and non-steroid binding activity or non-steroid sensitivityof the Group H ligand binding domain. Preferably, the isolatedpolynucleotide comprises a codon mutation that results in a substitutionof an amino acid residue at a position equivalent or analogous to a)amino acid residue 21, 48, 51, 59, 62, 93, 95, 109, 120, 123, 125, 218,219, 223, 230, 234, or 238 of SEQ ID NO: 1, b) amino acid residues 95and 110 of SEQ ID NO: 1, c) amino acid residues 218 and 219 of SEQ IDNO: 1, d) amino acid residue 121, 213, or 217 of SEQ ID NO: 2, or e)amino acid residue 105 of SEQ ID NO: 3. More preferably, the isolatedpolynucleotide comprises a codon mutation that results in a substitutionof a) an alanine residue at a position equivalent or analogous to aminoacid residue 21, 48, 51, 59, 62, 93, 95, 109, 120, 125, 218, 219, 223,230, 234, or 238 of SEQ ID NO: 1, b) a phenylalanine residue at aposition equivalent or analogous to amino acid residue 123 of SEQ ID NO:1, c) an alanine residue at a position equivalent or analogous to aminoacid residue 95 of SEQ ID NO: 1 and a proline residue at a positionequivalent or analogous to amino acid residue 110 of SEQ ID NO: 1, d) analanine residue at a position equivalent or analogous to amino acidresidues 218 and 219 of SEQ ID NO: 1, e) an arginine or a leucineresidue at a position equivalent or analogous to amino acid residue 121of SEQ ID NO: 2, f) an alanine residue at a position equivalent oranalogous to amino acid residue 213 of SEQ ID NO: 2, g) an alanine or aserine residue at a position equivalent or analogous to amino acidresidue 217 of SEQ ID NO: 2, or h) a proline residue at a positionequivalent or analogous to amino acid residue 105 of SEQ ID NO: 3. Evenmore preferably, the isolated polynucleotide comprises a codon mutationthat results in a substitution mutation of Q21A, F48A, I51A, V59A, I62A,M93A, R95A, F109A, Y120A, A123F, M125A, M218A, C219A, L223A, L230A,L234A, W238A, R95A/A110P, or M218A/C219A of SEQ ID NO: 1, b) G121R,G121L, N213A, C217A, or C217S of SEQ ID NO: 2, or c) A105P of SEQ ID NO:3.

In addition, the present invention also relates to an isolatedpolynucleotide encoding a Group H nuclear receptor ligand binding domaincomprising a substitution mutation, wherein the substitution mutationenhances ligand binding activity or ligand sensitivity of the Group Hnuclear receptor ligand binding domain.

In a specific embodiment, the present invention relates to an isolatedpolynucleotide encoding a Group H nuclear receptor ligand binding domaincomprising a substitution mutation, wherein the substitution mutationenhances steroid binding activity or steroid sensitivity of the Group Hnuclear receptor ligand binding domain. Preferably, the isolatedpolynucleotide comprises a codon mutation that results in a substitutionof an amino acid residue at a position equivalent or analogous to a)amino acid residue 52 or 96 of SEQ ID NO: 1 or b) amino acid residue 91of SEQ ID NO: 3. More preferably, the isolated polynucleotide comprisesa codon mutation that results in a substitution of a) a leucine, valine,or isoleucine residue at a position equivalent or analogous to aminoacid residue 52 of SEQ ID NO: 1, b) an alanine, threonine, asparticacid, or methionine residue at a position equivalent or analogous toamino acid residue 96 of SEQ ID NO: 1, c) a threonine residue at aposition equivalent or analogous to amino acid residue 96 of SEQ ID NO:1, an isoleucine residue at a position equivalent or analogous to aminoacid residue 107 of SEQ ID NO: 1, and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, or d)an alanine residue at a position equivalent or analogous to amino acidresidue 91 of SEQ ID NO: 3. Even more preferably, the isolatedpolynucleotide comprises a codon mutation that results in a substitutionmutation of a) T52L, T52V, T52I, V96A, V96T, V96D, or V96M of SEQ ID NO:1 or b) G91A of SEQ ID NO: 3.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation enhances non-steroid binding activity ornon-steroid sensitivity of the Group H nuclear receptor ligand bindingdomain. Preferably, the isolated polynucleotide comprises a codonmutation that results in a substitution of an amino acid residue at aposition equivalent or analogous to amino acid residue 52, 55, or 96 ofSEQ ID NO: 1 or b) amino acid residue 91 of SEQ ID NO: 3. Morepreferably, the isolated polynucleotide comprises a codon mutation thatresults in a substitution of a) an alanine, valine, or isoleucineresidue at a position equivalent or analogous to amino acid residue 52of SEQ ID NO: 1, b) an alanine residue at a position equivalent oranalogous to amino acid residue 55 of SEQ ID NO: 1, c) an aspartic acidor methionine residue at a position equivalent or analogous to aminoacid residue 96 of SEQ ID NO: 1, or d) an alanine residue at a positionequivalent or analogous to amino acid residue 91 of SEQ ID NO: 3. Evenmore preferably, the isolated polynucleotide comprises a codon mutationthat results in a substitution mutation of a) T52A, T52V, T52I, T55A,V96D, or V96M of SEQ ID NO: 1 or b) G91A of SEQ ID NO: 3.

In another specific embodiment, the present invention relates to anisolated polynucleotide encoding a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, wherein thesubstitution mutation enhances both steroid binding activity or steroidsensitivity and non-steroid binding activity or non-steroid sensitivityof the Group H ligand binding domain. Preferably, the isolatedpolynucleotide comprises a codon mutation that results in a substitutionof an amino acid residue at a position equivalent or analogous to a)amino acid residue 52, 96, 107 or 175 of SEQ ID NO: 1, b) amino acidresidues 107 and 175 of SEQ ID NO: 1, c) amino acid residues 127 and 175of SEQ ID NO: 1, d) amino acid residues 107 and 127 of SEQ ID NO: 1, e)amino acid residues 107, 127 and 175 of SEQ ID NO: 1, f) amino acidresidues 52, 107 and 175 of SEQ ID NO: 1, g) amino acid residues 96, 107and 175 of SEQ ID NO: 1, or h) amino acid residue 91 of SEQ ID NO: 3.More preferably, the isolated polynucleotide comprises a codon mutationthat results in a substitution of a) a valine or isoleucine residue at aposition equivalent or analogous to amino acid residue 52 of SEQ ID NO:1, b) an aspartic acid or methionine residue at a position equivalent oranalogous to amino acid residue 96 of SEQ ID NO: 1, c) an isoleucineresidue at a position equivalent or analogous to amino acid residue 107of SEQ ID NO: 1, d) a glutamine residue at a position equivalent oranalogous to amino acid residues 175, e) an isoleucine residue at aposition equivalent or analogous to amino acid residue 107 of SEQ ID NO:1 and a glutamine residue at a position equivalent or analogous to aminoacid residue 175 of SEQ ID NO: 1, f) a glutamine residue at a positionequivalent or analogous to amino acid residues 127 and 175 of SEQ ID NO:1, g) an isoleucine residue at a position equivalent or analogous toamino acid residue 107 of SEQ ID NO: 1 and a glutamine residue at aposition equivalent or analogous to amino acid residue 127 of SEQ ID NO:1, h) an isoleucine residue at a position equivalent or analogous toamino acid residue 107 of SEQ ID NO: 1 and a glutamine residue at aposition equivalent or analogous to amino acid residues 127 and 175 ofSEQ ID NO: 1, i) a valine residue at a position equivalent or analogousto amino acid residue 52 of SEQ ID NO: 1, an isoleucine residue at aposition equivalent or analogous to amino acid residue 107 of SEQ ID NO:1 and a glutamine residue at a position equivalent or analogous to aminoacid residue 175 of SEQ ID NO: 1, j) an alanine residue at a positionequivalent or analogous to amino acid residue 96 of SEQ ID NO: 1, anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1 and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, k) analanine residue at a position equivalent or analogous to amino acidresidue 52 of SEQ ID NO: 1, an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1, and aglutamine residue at a position equivalent or analogous to amino acidresidue 175 of SEQ ID NO: 1, or l) an alanine residue at a positionequivalent or analogous to amino acid residue 91 of SEQ ID NO: 3. Evenmore preferably, the isolated polynucleotide comprises a codon mutationthat results in a substitution mutation of a) T52V, T52I, V96D, V96M,V107I, R175E, V107I/R175E, Y127E/R175E, V107I/Y127E, V107I/Y127E/R175E,T52V/V107I/R175E, V96A/V107I/R175E or T52A/V107I/R175E of SEQ ID NO: 1or b) G91A of SEQ ID NO: 3.

In addition, the present invention relates to an expression vectorcomprising a polynucleotide according the invention, operatively linkedto a transcription regulatory element. Preferably, the polynucleotideencoding a nuclear receptor ligand binding domain comprising asubstitution mutation is operatively linked with an expression controlsequence permitting expression of the nuclear receptor ligand bindingdomain in an expression competent host cell. The expression controlsequence may comprise a promoter that is functional in the host cell inwhich expression is desired. The vector may be a plasmid DNA molecule ora viral vector. Preferred viral vectors include retrovirus, adenovirus,adeno-associated virus, herpes virus, and vaccinia virus. The inventionfurther relates to a replication defective recombinant virus comprisingin its genome, the polynucleotide encoding a nuclear receptor ligandbinding domain comprising a substitution mutation as described above.Thus, the present invention also relates to an isolated host cellcomprising such an expression vector, wherein the transcriptionregulatory element is operative in the host cell.

The present invention also relates to an isolated polypeptide encoded bya polynucleotide according to the invention.

Polypeptides of the Invention

The novel nuclear receptor-based inducible gene expression system of theinvention comprises at least one gene expression cassette comprising apolynucleotide that encodes a polypeptide comprising a Group H nuclearreceptor ligand binding domain comprising a substitution mutation. Thus,the present invention also provides an isolated polypeptide comprising aGroup H nuclear receptor ligand binding domain comprising a substitutionmutation according to the invention.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprises a substitution mutation at a positionequivalent or analogous to a) amino acid residue 20, 21, 48, 51, 52, 55,58, 59, 61, 62, 92, 93, 95, 96, 107, 109, 110, 120, 123, 125, 175, 218,219, 223, 230, 234, or 238 of SEQ ID NO: 1, b) amino acid residues 95and 110 of SEQ ID NO: 1, c) amino acid residues 218 and 219 of SEQ IDNO: 1, d) amino acid residues 107 and 175 of SEQ ID NO: 1, e) amino acidresidues 127 and 175 of SEQ ID NO: 1, f) amino acid residues 107 and 127of SEQ ID NO: 1, g) amino acid residues 107, 127 and 175 of SEQ ID NO:1, h) amino acid residues 52, 107 and 175 of SEQ ID NO: 1, i) amino acidresidues 96, 107 and 175 of SEQ ID NO: 1, j) amino acid residues 107,110 and 175 of SEQ ID NO: 1, k) amino acid residue 107, 121, 213, or 217of SEQ ID NO: 2, or l) amino acid residue 91 or 105 of SEQ ID NO: 3. Ina preferred embodiment, the Group H nuclear receptor ligand bindingdomain is from an ecdysone receptor.

Preferably, the Group H nuclear receptor ligand binding domain comprisesa substitution of a) an alanine residue at a position equivalent oranalogous to amino acid residue 20, 21, 48, 51, 55, 58, 59, 61, 62, 92,93, 95, 109, 120, 125, 218, 219, 223, 230, 234, or 238 of SEQ ID NO: 1,b) an alanine, valine, isoleucine, or leucine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, c) analanine, threonine, aspartic acid, or methionine residue at a positionequivalent or analogous to amino acid residue 96 of SEQ ID NO: 1, d) aproline, serine, methionine, or leucine residue at a position equivalentor analogous to amino acid residue 110 of SEQ ID NO: 1, e) aphenylalanine residue at a position equivalent or analogous to aminoacid residue 123 of SEQ ID NO: 1, f) an alanine residue at a positionequivalent or analogous to amino acid residue 95 of SEQ ID NO: 1 and aproline residue at a position equivalent or analogous to amino acidresidue 110 of SEQ ID NO: 1, g) an alanine residue at a positionequivalent or analogous to amino acid residues 218 and 219 of SEQ ID NO:1, h) an isoleucine residue at a position equivalent or analogous toamino acid residue 107 of SEQ ID NO: 1, i) a glutamine residue at aposition equivalent or analogous to amino acid residues 175, j) anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1 and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, k) aglutamine residue at a position equivalent or analogous to amino acidresidues 127 and 175 of SEQ ID NO: 1, l) an isoleucine residue at aposition equivalent or analogous to amino acid residue 107 of SEQ ID NO:1 and a glutamine residue at a position equivalent or analogous to aminoacid residue 127 of SEQ ID NO: 1, m) an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamine residue at a position equivalent or analogous to amino acidresidues 127 and 175 of SEQ ID NO: 1, n) a valine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1 and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, o) analanine residue at a position equivalent or analogous to amino acidresidue 96 of SEQ ID NO: 1, an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamine residue at a position equivalent or analogous to amino acidresidue 175 of SEQ ID NO: 1, p) an alanine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1, and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, q) athreonine residue at a position equivalent or analogous to amino acidresidue 96 of SEQ ID NO: 1, an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1, and aglutamine residue at a position equivalent or analogous to amino acidresidue 175 of SEQ ID NO: 1, r) an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1, aproline at a position equivalent or analogous to amino acid 110 of SEQID NO: 1, and a glutamine residue at a position equivalent or analogousto amino acid residue 175 of SEQ ID NO: 1, s) a proline at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 2, t) anarginine or a leucine at a position equivalent or analogous to aminoacid residue 121 of SEQ ID NO: 2, u) an alanine at a position equivalentor analogous to amino acid residue 213 of SEQ ID NO: 2, v) an alanine ora serine at a position equivalent or analogous to amino acid residue 217of SEQ ID NO: 2, w) an alanine at a position equivalent or analogous toamino acid residue 91 of SEQ ID NO: 3, or x) a proline at a positionequivalent or analogous to amino acid residue 105 of SEQ ID NO: 3. In apreferred embodiment, the Group H nuclear receptor ligand binding domainis from an ecdysone receptor.

In another specific embodiment, the Group H nuclear receptor ligandbinding domain comprising a substitution mutation is an ecdysonereceptor ligand binding domain polypeptide comprising a substitutionmutation, wherein the substitution mutation is selected from the groupconsisting of a) E20A, Q21A, F48A, I51A, T52A, T52V, T52I, T52L, T55A,T58A, V59A, L61A, I62A, M92A, M93A, R95A, V96A, V96T, V96D, V96M, V107I,F109A, A110P, A110S, A110M, A110L, Y120A, A123F, M125A, R175E, M218A,C219A, L223A, L230A, L234A, W238A, R95A/A110P, M218A/C219A, V107I/R175E,Y127E/R175E, V107I/Y127E, V107I/Y127E/R175E, T52V/V107I/R175E,V96A/V107I/R175E, T52A/V107I/R175E V96T/V107I/R175E, orV107I/A110P/R175E substitution mutation of SEQ ID NO: 1, b) A107P,G121R, G121L, N213A, C217A, or C217S substitution mutation of SEQ ID NO:2, and c) G91A or A105P substitution mutation of SEQ ID NO: 3.

The present invention also provides an isolated polypeptide selectedfrom the group consisting of a) an isolated polypeptide comprising atransactivation domain, a DNA-binding domain, and a Group H nuclearreceptor ligand binding domain comprising a substitution mutationaccording to the invention; b) an isolated polypeptide comprising aDNA-binding domain and a Group H nuclear receptor ligand binding domaincomprising a substitution mutation according to the invention; and c) anisolated polypeptide comprising a transactivation domain and a Group Hnuclear receptor ligand binding domain comprising a substitutionmutation according to the invention. In a preferred embodiment, theGroup H nuclear receptor ligand binding domain is from an ecdysonereceptor.

The present invention also provides an isolated hybrid polypeptideselected from the group consisting of a) an isolated hybrid polypeptidecomprising a transactivation domain, a DNA-binding domain, and a Group Hnuclear receptor ligand binding domain comprising a substitutionmutation according to the invention; b) an isolated hybrid polypeptidecomprising a DNA-binding domain and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation according to theinvention; and c) an isolated hybrid polypeptide comprising atransactivation domain and a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation according to the invention. Ina preferred embodiment, the Group H nuclear receptor ligand bindingdomain is from an ecdysone receptor.

The present invention also provides an isolated polypeptide comprising aGroup H nuclear receptor ligand binding domain comprising a substitutionmutation that affects ligand binding activity or ligand sensitivity ofthe Group H nuclear receptor ligand binding domain.

In particular, the present invention relates to an isolated Group Hnuclear receptor polypeptide comprising a ligand binding domaincomprising a substitution mutation that reduces ligand binding activityor ligand sensitivity of the Group H nuclear receptor ligand bindingdomain.

In a specific embodiment, the present invention relates to an isolatedpolypeptide comprising a Group H nuclear receptor ligand binding domaincomprising a substitution mutation that reduces steroid binding activityor steroid sensitivity of the Group H nuclear receptor ligand bindingdomain. Preferably, the isolated polypeptide comprises a substitution ofan amino acid residue at a position equivalent or analogous to a) aminoacid residue 20, 21, 48, 51, 52, 55, 58, 59, 62, 92, 93, 95, 109, 110,120, 123, 125, 218, 219, 223, 230, 234, or 238 of SEQ ID NO: 1, b) aminoacid residue 107, 121, 213, or 217 of SEQ ID NO: 2, or c) amino acidresidue 105 of SEQ ID NO: 3. More preferably, the isolated polypeptidecomprises a substitution of a) an alanine residue at a positionequivalent or analogous to amino acid residue 20, 21, 48, 51, 52, 55,58, 59, 62, 92, 93, 95, 109, 120, 125, 218, 219, 223, 230, 234, or 238of SEQ ID NO: 1, b) a proline residue at a position equivalent oranalogous to amino acid residue 110 of SEQ ID NO: 1, c) a phenylalanineresidue at a position equivalent or analogous to amino acid residue 123of SEQ ID NO: 1, d) an alanine residue at a position equivalent oranalogous to amino acid residue 95 of SEQ ID NO: 1 and a proline residueat a position equivalent or analogous to amino acid residue 110 of SEQID NO: 1, e) an alanine residue at a position equivalent or analogous toamino acid residues 218 and 219 of SEQ ID NO: 1, f) a proline residue ata position equivalent or analogous to amino acid residue 107 of SEQ IDNO: 2, g) an arginine or leucine residue at a position equivalent oranalogous to amino acid residue 121 of SEQ ID NO: 2, h) an alanineresidue at a position equivalent or analogous to amino acid residue 213of SEQ ID NO: 2, i) an alanine or a serine residue at a positionequivalent or analogous to amino acid residue 217 of SEQ ID NO: 2, or j)a proline residue at a position equivalent or analogous to amino acidresidue 105 of SEQ ID NO: 3. Even more preferably, the isolatedpolypeptide comprises a substitution mutation of a) E20A, Q21A, F48A,I51A, T52A, T55A, T58A, V59A, I62A, M92A, M93A, R95A, F109A, A110P,Y120A, A123F, M125A, M218A, C219A, L223A, L230A, L234A, W238A,R95A/A110P, or M218A/C219A of SEQ ID NO: 1, b) A107P, G121R, G121L,N213A, C217A, or C217S of SEQ ID NO: 2, or c) A105P of SEQ ID NO: 3.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation that eliminatessteroid binding activity or steroid sensitivity of the Group H ligandbinding domain. Preferably, the isolated polypeptide comprises asubstitution of an amino acid residue at a position equivalent oranalogous to a) amino acid residue 58 or 110 of SEQ ID NO: 1, b) aminoacid residues 107, 110 and 175 of SEQ ID NO: 1, c) amino acid residue107, 121, 213, or 217 of SEQ ID NO: 2, or d) amino acid residue 105 ofSEQ ID NO: 3. More preferably, the isolated polynucleotide comprises acodon mutation that results in a substitution of a) an alanine at aposition equivalent or analogous to amino acid residue 58 of SEQ ID NO:1, b) a proline, leucine, serine, or methionine residue at a positionequivalent or analogous to amino acid residue 110 of SEQ ID NO: 1, c) anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1, a proline residue at a position equivalentor analogous to amino acid 110 of SEQ ID NO: 1, and a glutamine residueat a position equivalent or analogous to amino acid residue 175 of SEQID NO: 1, d) a proline at a position equivalent or analogous to aminoacid residue 107 of SEQ ID NO: 2, e) an arginine or a leucine at aposition equivalent or analogous to amino acid residue 121 of SEQ ID NO:2, f) an alanine at a position equivalent or analogous to amino acidresidue 213 of SEQ ID NO: 2, g) an alanine or a serine at a positionequivalent or analogous to amino acid residue 217 of SEQ ID NO: 2, or h)a proline at a position equivalent or analogous to amino acid residue105 of SEQ ID NO: 3. Even more preferably, the isolated polypeptidecomprises a substitution mutation selected from the group consisting ofa) T58A, A110P, A110L, A110S, A110M, or V107I/A110P/R175E substitutionmutation of SEQ ID NO: 1, b) A107P, G121R, G121L, N213A, C217A, or C217Ssubstitution mutation of SEQ ID NO: 2, and c) A105P substitutionmutation of SEQ ID NO: 3.

The present invention also relates to an isolated polypeptide comprisingan ecdysone receptor ligand binding domain comprising a substitutionmutation, wherein the ecdysone receptor ligand binding domain lackssteroid binding activity. Preferably, the ecdysone receptor ligandbinding domain comprises a substitution mutation at an equivalent oranalogous amino acid residue to a) amino acid residue 58 or 110 of SEQID NO: 1, b) amino acid residues 107, 110 and 175 of SEQ ID NO: 1, c)amino acid residue 107, 121, 213, or 217 of SEQ ID NO: 2, or d) aminoacid residue 105 of SEQ ID NO: 3. More preferably, the ecdysone receptorligand binding domain comprises a substitution of a) an alanine at aposition equivalent or analogous to amino acid residue 58 of SEQ ID NO:1, b) a proline, leucine, serine, or methionine residue at a positionequivalent or analogous to amino acid residue 110 of SEQ ID NO: 1, c) anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1, a proline residue at a position equivalentor analogous to amino acid residue 110 of SEQ ID NO: 1, and a glutamineresidue at a position equivalent or analogous to amino acid residue 175of SEQ ID NO: 1, d) a proline at a position equivalent or analogous toamino acid residue 107 of SEQ ID NO: 2, e) an arginine or a leucine at aposition equivalent or analogous to amino acid residue 121 of SEQ ID NO:2, f) an alanine at a position equivalent or analogous to amino acidresidue 213 of SEQ ID NO: 2, g) an alanine or a serine at a positionequivalent or analogous to amino acid residue 217 of SEQ ID NO: 2, or h)a proline at a position equivalent or analogous to amino acid residue105 of SEQ ID NO: 3. Even more preferably, the ecdysone receptor ligandbinding domain comprises a substitution mutation selected from the groupconsisting of a) T58A, A110P, A110L, A110S, A110M, or V107I/A110P/R175Esubstitution mutation of SEQ ID NO: 1, b) A107P, G121R, G121L, N213A,C217A, or C217S substitution mutation of SEQ ID NO: 2, and c) A105Psubstitution mutation of SEQ ID NO: 3. In a specific embodiment, theecdysone receptor ligand binding domain lacks steroid binding activityselected from the group consisting of ecdysone binding activity,20-hydroxyecdysone binding activity, ponasterone A binding activity, andmuristerone A binding activity.

In another specific embodiment, the isolated polypeptide comprising anecdysone receptor ligand binding domain comprising a substitutionmutation, wherein the ecdysone receptor ligand binding domain lackssteroid binding activity and is encoded by a polynucleotide thathybridizes to a polynucleotide comprising a codon mutation that resultsin a substitution mutation selected from the group consisting of a)T58A, A110P, A110L, A110S, A110M, or V107I/A110P/R175E substitutionmutation of SEQ ID NO: 1, b) A107P, G121R, G121L, N213A, C217A, or C217Ssubstitution mutation of SEQ ID NO: 2, and c) A105P substitutionmutation of SEQ ID NO: 3 under hybridization conditions comprising ahybridization step in less than 500 mM salt and at least 37 degreesCelsius, and a washing step in 2×SSPE at at least 63 degrees Celsius. Ina preferred embodiment, the hybridization conditions comprise less than200 mM salt and at least 37 degrees Celsius for the hybridization step.In another preferred embodiment, the hybridization conditions comprise2×SSPE and 63 degrees Celsius for both the hybridization and washingsteps. In another preferred embodiment, the ecdysone receptor ligandbinding domain lacks steroid binding activity selected from the groupconsisting of ecdysone binding activity, 20-hydroxyecdysone bindingactivity, ponasterone A binding activity, and muristerone A bindingactivity.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation that reducesnon-steroid binding activity or non-steroid sensitivity of the Group Hnuclear receptor ligand binding domain. Preferably, the isolatedpolypeptide comprises a substitution of an amino acid residue at aposition equivalent or analogous to amino acid residue a) 21, 48, 51,52, 59, 62, 93, 95, 96, 109, 120, 123, 125, 218, 219, 223, 230, 234, or238 of SEQ ID NO: 1, b) 121, 213, or 217 of SEQ ID NO: 2, or c) 105 ofSEQ ID NO: 3. More preferably, the isolated polypeptide comprises asubstitution of a) an alanine residue at a position equivalent oranalogous to amino acid residue 21, 48, 51, 59, 62, 93, 95, 96, 109,120, 125, 218, 219, 223, 230, 234, or 238 of SEQ ID NO: 1, b) a leucineresidue at at a position equivalent or analogous to amino acid residue52 of SEQ ID NO: 1, c) a threonine residue at at a position equivalentor analogous to amino acid residue 96 of SEQ ID NO: 1, d) aphenylalanine residue at at a position equivalent or analogous to aminoacid residue 123 of SEQ ID NO: 1, e) an alanine residue at at a positionequivalent or analogous to amino acid residue 95 of SEQ ID NO: 1 and aproline residue at a position equivalent or analogous to amino acidresidue 110 of SEQ ID NO: 1, f) an alanine residue at at a positionequivalent or analogous to amino acid residues 218 and 219 of SEQ ID NO:1, g) an arginine or a leucine residue at a position equivalent oranalogous to amino acid residue 121 of SEQ ID NO: 2, h) an alanineresidue at a position equivalent or analogous to amino acid residue 213of SEQ ID NO: 2, i) an alanine or a serine residue at a positionequivalent or analogous to amino acid residue 217 of SEQ ID NO: 2, or j)a proline residue at a position equivalent or analogous to amino acidresidue 105 of SEQ ID NO: 3. Even more preferably, the isolatedpolypeptide comprises a substitution mutation of a) Q21A, F48A, I51A,T52L, V59A, I62A, M93A, R95A, V96A, V96T, F109A, Y120A, A123F, M125A,M218A, C219A, L223A, L230A, L234A, W238A, R95A/A110P, or M218/C219A ofSEQ ID NO: 1, b) G121R, G121L, N213A, C217A, or C217S of SEQ ID NO: 2,or c) A105P of SEQ ID NO: 3.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor polypeptideligand binding domain comprising a substitution mutation that eliminatesnon-steroid binding activity or non-steroid sensitivity of the Group Hligand binding domain.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor polypeptideligand binding domain comprising a substitution mutation that reducesboth steroid binding activity or steroid sensitivity and non-steroidbinding activity or non-steroid sensitivity of the Group H ligandbinding domain. Preferably, the isolated polypeptide comprises asubstitution of an amino acid residue at a position equivalent oranalogous to a) amino acid residue 21, 48, 51, 59, 62, 93, 95, 109, 120,123, 125, 218, 219, 223, 230, 234, or 238 of SEQ ID NO: 1, b) amino acidresidues 95 and 110 of SEQ ID NO: 1, c) amino acid residues 218 and 219of SEQ ID NO: 1, d) amino acid residue 121, 213, or 217 of SEQ ID NO: 2,or e) amino acid residue 105 of SEQ ID NO: 3. More preferably, theisolated polypeptide comprises a substitution of a) an alanine residueat a position equivalent or analogous to amino acid residue 21, 48, 51,59, 62, 93, 95, 109, 120, 125, 218, 219, 223, 230, 234, or 238 of SEQ IDNO: 1, b) a phenylalanine residue at a position equivalent or analogousto amino acid residue 123 of SEQ ID NO: 1, c) an alanine residue at aposition equivalent or analogous to amino acid residue 95 of SEQ ID NO:1 and a proline residue at a position equivalent or analogous to aminoacid residue 110 of SEQ ID NO: 1, d) an alanine residue at a positionequivalent or analogous to amino acid residues 218 and 219 of SEQ ID NO:1, e) an arginine or leucine residue at a position equivalent oranalogous to amino acid residue 121 of SEQ ID NO: 2, f) an alanineresidue at a position equivalent or analogous to amino acid residue 213of SEQ ID NO: 2, g) an alanine or serine residue at a positionequivalent or analogous to amino acid residue 217 of SEQ ID NO: 2, or h)a proline residue at a position equivalent or analogous to amino acidresidue 105 of SEQ ID NO: 3. Even more preferably, the isolatedpolypeptide comprises a substitution mutation of Q21A, F48A, I51A, V59A,I62A, M93A, R95A, F109A, Y120A, A123F, M125A, M218A, C219A, L223A,L230A, L234A, W238A, R95A/A110P, or M218A/C219A of SEQ ID NO: 1, b)G121R, G121L, N213A, C217A, or C217S of SEQ ID NO: 2, or c) A105P of SEQID NO: 3.

In addition, the present invention also relates to an isolatedpolypeptide comprising a Group H nuclear receptor ligand binding domaincomprising a substitution mutation that enhances ligand binding activityor ligand sensitivity of the Group H nuclear receptor ligand bindingdomain.

In a specific embodiment, the present invention relates to an isolatedpolypeptide comprising a Group H nuclear receptor ligand binding domaincomprising a substitution mutation that enhances steroid bindingactivity or steroid sensitivity of the Group H nuclear receptor ligandbinding domain. Preferably, the isolated polypeptide comprises asubstitution of an amino acid residue at a position equivalent oranalogous to a) amino acid residue 52 or 96 of SEQ ID NO: 1, b) aminoacid residues 96, 107 and 175 of SEQ ID NO: 1, or c) amino acid residue91 of SEQ ID NO: 3. More preferably, the isolated polypeptide comprisesa substitution of a) a leucine, valine, or isoleucine residue at aposition equivalent or analogous to amino acid residue 52 of SEQ ID NO:1, b) an alanine, threonine, aspartic acid, or methionine residue at aposition equivalent or analogous to amino acid residue 96 of SEQ ID NO:1, c) a threonine residue at a position equivalent or analogous to aminoacid residue 96 of SEQ ID NO: 1, an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamine residue at a position equivalent or analogous to amino acidresidue 175 of SEQ ID NO: 1, or d) an alanine residue at a positionequivalent or analogous to amino acid residue 91 of SEQ ID NO: 3. Evenmore preferably, the isolated polypeptide comprises a substitutionmutation of a) T52L, T52V, T52I, V96A, V96T, V96D, V96M, orV96T/V107I/R175E of SEQ ID NO: 1 or b) G91A of SEQ ID NO: 3.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation that enhancesnon-steroid binding activity or non-steroid sensitivity of the Group Hnuclear receptor ligand binding domain. Preferably, the isolatedpolypeptide comprises a substitution of an amino acid residue at aposition equivalent or analogous to amino acid residue 52, 55, or 96 ofSEQ ID NO: 1 or b) amino acid residue 91 of SEQ ID NO: 3. Morepreferably, the isolated polypeptide comprises a substitution of a) analanine, valine, or isoleucine residue at a position equivalent oranalogous to amino acid residue 52 of SEQ ID NO: 1, b) an alanineresidue at a position equivalent or analogous to amino acid residue 55of SEQ ID NO: 1, c) an aspartic acid or methionine residue at a positionequivalent or analogous to amino acid residue 96 of SEQ ID NO: 1, or d)an alanine residue at a position equivalent or analogous to amino acidresidue 91 of SEQ ID NO: 3. Even more preferably, the isolatedpolypeptide comprises a substitution mutation of a) T52A, T52V, T52I,T55A, V96D, or V96M of SEQ ID NO: 1 or b) G91A of SEQ ID NO: 3.

In another specific embodiment, the present invention relates to anisolated polypeptide comprising a Group H nuclear receptor ligandbinding domain comprising a substitution mutation that enhances bothsteroid binding activity or steroid sensitivity and non-steroid bindingactivity or non-steroid sensitivity of the Group H ligand bindingdomain. Preferably, the isolated polypeptide comprises a substitution ofan amino acid residue at a position equivalent or analogous to a) aminoacid residue 52, 96, 107 or 175 of SEQ ID NO: 1, b) amino acid residues107 and 175 of SEQ ID NO: 1, c) amino acid residues 127 and 175 of SEQID NO: 1, d) amino acid residues 107 and 127 of SEQ ID NO: 1, e) aminoacid residues 107, 127 and 175 of SEQ ID NO: 1, f) amino acid residues52, 107 and 175 of SEQ ID NO: 1, g) amino acid residues 96, 107 and 175of SEQ ID NO: 1, or h) amino acid residue 91 of SEQ ID NO: 3. Morepreferably, the isolated polypeptide comprises a substitution of a) avaline or isoleucine residue at a position equivalent or analogous toamino acid residue 52 of SEQ ID NO: 1, b) an aspartic acid or methionineresidue at a position equivalent or analogous to amino acid residue 96of SEQ ID NO: 1, c) an isoleucine residue at a position equivalent oranalogous to amino acid residue 107 of SEQ ID NO: 1, d) a glutamineresidue at a position equivalent or analogous to amino acid residues175, e) an isoleucine residue at a position equivalent or analogous toamino acid residue 107 of SEQ ID NO: 1 and a glutamine residue at aposition equivalent or analogous to amino acid residue 175 of SEQ ID NO:1, f) a glutamine residue at a position equivalent or analogous to aminoacid residues 127 and 175 of SEQ ID NO: 1, g) an isoleucine residue at aposition equivalent or analogous to amino acid residue 107 of SEQ ID NO:1 and a glutamine residue at a position equivalent or analogous to aminoacid residue 127 of SEQ ID NO: 1, h) an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamine residue at a position equivalent or analogous to amino acidresidues 127 and 175 of SEQ ID NO: 1, i) a valine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1 and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, j) analanine residue at a position equivalent or analogous to amino acidresidue 96 of SEQ ID NO: 1, an isoleucine residue at a positionequivalent or analogous to amino acid residue 107 of SEQ ID NO: 1 and aglutamine residue at a position equivalent or analogous to amino acidresidue 175 of SEQ ID NO: 1, k) an alanine residue at a positionequivalent or analogous to amino acid residue 52 of SEQ ID NO: 1, anisoleucine residue at a position equivalent or analogous to amino acidresidue 107 of SEQ ID NO: 1, and a glutamine residue at a positionequivalent or analogous to amino acid residue 175 of SEQ ID NO: 1, or l)an alanine residue at a position equivalent or analogous to amino acidresidue 91 of SEQ ID NO: 3. Even more preferably, the isolatedpolypeptide comprises a substitution mutation of a) T52V, T521, V96D,V96M, V107I, R175E, V107I/R175E, Y127E/R175E, V107I/Y127E,V107I/Y127E/R175E, T52V/V107I/R175E, V96A/V107I/R175E, orT52A/V107I/R175E of SEQ ID NO: 1 or b) G91A of SEQ ID NO: 3.

The present invention also relates to compositions comprising anisolated polypeptide according to the invention.

Method of Modulating Gene Expression of the Invention

Applicants' invention also relates to methods of modulating geneexpression in a host cell using a gene expression modulation systemaccording to the invention. Specifically, Applicants' invention providesa method of modulating the expression of a gene in a host cellcomprising the steps of: a) introducing into the host cell a geneexpression modulation system according to the invention; and b)introducing into the host cell a ligand; wherein the gene to bemodulated is a component of a gene expression cassette comprising: i) aresponse element comprising a domain recognized by the DNA bindingdomain of the gene expression system; ii) a promoter that is activatedby the transactivation domain of the gene expression system; and iii) agene whose expression is to be modulated, whereby upon introduction ofthe ligand into the host cell, expression of the gene is modulated.

The invention also provides a method of modulating the expression of agene in a host cell comprising the steps of: a) introducing into thehost cell a gene expression modulation system according to theinvention; b) introducing into the host cell a gene expression cassetteaccording to the invention, wherein the gene expression cassettecomprises i) a response element comprising a domain recognized by theDNA binding domain from the gene expression system; ii) a promoter thatis activated by the transactivation domain of the gene expressionsystem; and iii) a gene whose expression is to be modulated; and c)introducing into the host cell a ligand; whereby upon introduction ofthe ligand into the host cell, expression of the gene is modulated.

Applicants' invention also provides a method of modulating theexpression of a gene in a host cell comprising a gene expressioncassette comprising a response element comprising a domain to which theDNA binding domain from the first hybrid polypeptide of the geneexpression modulation system binds; a promoter that is activated by thetransactivation domain of the second hybrid polypeptide of the geneexpression modulation system; and a gene whose expression is to bemodulated; wherein the method comprises the steps of: a) introducinginto the host cell a gene expression modulation system according to theinvention; and b) introducing into the host cell a ligand; whereby uponintroduction of the ligand into the host, expression of the gene ismodulated.

Genes of interest for expression in a host cell using Applicants'methods may be endogenous genes or heterologous genes. Nucleic acid oramino acid sequence information for a desired gene or protein can belocated in one of many public access databases, for example, GENBANK,EMBL, Swiss-Prot, and PIR, or in many biology related journalpublications. Thus, those skilled in the art have access to nucleic acidsequence information for virtually all known genes. Such information canthen be used to construct the desired constructs for the insertion ofthe gene of interest within the gene expression cassettes used inApplicants' methods described herein.

Examples of genes of interest for expression in a host cell usingApplicants' methods include, but are not limited to: antigens producedin plants as vaccines, enzymes like alpha-amylase, phytase, glucanes,xylase and xylanase, genes for resistance against insects, nematodes,fungi, bacteria, viruses, and abiotic stresses, nutraceuticals,pharmaceuticals, vitamins, genes for modifying amino acid content,herbicide resistance, cold, drought, and heat tolerance, industrialproducts, oils, protein, carbohydrates, antioxidants, male sterileplants, flowers, fuels, other output traits, genes encodingtherapeutically desirable polypeptides or products that may be used totreat a condition, a disease, a disorder, a dysfunction, a geneticdefect, such as monoclonal antibodies, enzymes, proteases, cytokines,interferons, insulin, erthropoietin, clotting factors, other bloodfactors or components, viral vectors for gene therapy, virus forvaccines, targets for drug discovery, functional genomics, andproteomics analyses and applications, and the like.

Acceptable ligands are any that modulate expression of the gene whenbinding of the DNA binding domain of the gene expression systemaccording to the invention to the response element in the presence ofthe ligand results in activation or suppression of expression of thegenes. Preferred ligands include an ecdysteroid, such as ecdysone,20-hydroxyecdysone, ponasterone A, muristerone A, and the like,9-cis-retinoic acid, synthetic analogs of retinoic acid,N,N′-diacylhydrazines such as those disclosed in U.S. Pat. Nos.6,013,836; 5,117,057; 5,530,028; and 5,378,726; dibenzoylalkylcyanohydrazines such as those disclosed in European Application No.461,809; N-alkyl-N,N′-diaroylhydrazines such as those disclosed in U.S.Pat. No. 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as thosedisclosed in European Application No. 234,994;N-aroyl-N-alkyl-N′-aroylhydrazines such as those described in U.S. Pat.No. 4,985,461; each of which is incorporated herein by reference andother similar materials including3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide,oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol,25-epoxycholesterol, T0901317,5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS),7-ketocholesterol-3-sulfate, farnesol, bile acids, 1,1-biphosphonateesters, Juvenile hormone III, and the like.

In a preferred embodiment, the ligand for use in Applicants' method ofmodulating expression of gene is a compound of the formula:

wherein:

-   -   E is a (C₄-C₆)alkyl containing a tertiary carbon or a        cyano(C₃-C₅)alkyl containing a tertiary carbon;    -   R¹ is H, Me, Et, i-Pr, F, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl,        CH₂OH, CH₂OMe, CH₂CN, CN, C^(o)CH, 1-propynyl, 2-propynyl,        vinyl, OH, OMe, OEt, cyclopropyl, CF₂CF₃, CH═CHCN, allyl, azido,        SCN, or SCHF₂;    -   R² is H, Me, Et, n-Pr, i-Pr, formyl, CF₃, CHF₂, CHCl₂, CH₂F,        CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C^(o)CH, 1-propynyl,        2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe₂,        NEt₂, SMe, SEt, SOCF₃, OCF₂CF₂H, COEt, cyclopropyl, CF₂CF₃,        CH═CHCN, allyl, azido, OCF₃, OCHF₂, O-i-Pr, SCN, SCHF₂, SOMe,        NH—CN, or joined with R³ and the phenyl carbons to which R² and        R³ are attached to form an ethylenedioxy, a dihydrofuryl ring        with the oxygen adjacent to a phenyl carbon, or a dihydropyryl        ring with the oxygen adjacent to a phenyl carbon;    -   R³ is H, Et, or joined with R² and the phenyl carbons to which        R² and R³ are attached to form an ethylenedioxy, a dihydrofuryl        ring with the oxygen adjacent to a phenyl carbon, or a        dihydropyryl ring with the oxygen adjacent to a phenyl carbon;    -   R⁴, R⁵, and R⁶ are independently H, Me, Et, F, Cl, Br, formyl,        CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CN, C^(o)CH, 1-propynyl,        2-propynyl, vinyl, OMe, OEt, SMe, or SEt.

In another preferred embodiment, the ligand for use in Applicants'method of modulating expression of gene is an ecdysone,20-hydroxyecdysone, ponasterone A, muristerone A, an oxysterol, a 22(R)hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol,T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS),7-ketocholesterol-3-sulfate, farnesol, bile acids, 1,1-biphosphonateesters, or Juvenile hormone III.

In another preferred embodiment, a second ligand may be used in additionto the first ligand discussed above in Applicants' method of modulatingexpression of a gene. Preferably, this second ligand is 9-cis-retinoicacid or a synthetic analog of retinoic acid.

Host Cells and Non-Human Organisms of the Invention

As described above, the gene expression modulation system of the presentinvention may be used to modulate gene expression in a host cell.Expression in transgenic host cells may be useful for the expression ofvarious genes of interest. Applicants' invention provides for modulationof gene expression in prokaryotic and eukaryotic host cells. Expressionin transgenic host cells is useful for the expression of variouspolypeptides of interest including but not limited to antigens producedin plants as vaccines, enzymes like alpha-amylase, phytase, glucanes,xylase and xylanase, genes for resistance against insects, nematodes,fungi, bacteria, viruses, and abiotic stresses, antigens,nutraceuticals, pharmaceuticals, vitamins, genes for modifying aminoacid content, herbicide resistance, cold, drought, and heat tolerance,industrial products, oils, protein, carbohydrates, antioxidants, malesterile plants, flowers, fuels, other output traits, therapeuticpolypeptides, pathway intermediates; for the modulation of pathwaysalready existing in the host for the synthesis of new productsheretofore not possible using the host; cell based assays; functionalgenomics assays, biotherapeutic protein production, proteomics assays,and the like. Additionally the gene products may be useful forconferring higher growth yields of the host or for enabling analternative growth mode to be utilized.

Thus, Applicants' invention provides an isolated host cell comprising agene expression system according to the invention. The present inventionalso provides an isolated host cell comprising a gene expressioncassette according to the invention. Applicants' invention also providesan isolated host cell comprising a polynucleotide or a polypeptideaccording to the invention. The present invention also relates to a hostcell transfected with an expression vector according to the invention.The host cell may be a bacterial cell, a fungal cell, a nematode cell,an insect cell, a fish cell, a plant cell, an avian cell, an animalcell, or a mammalian cell. In still another embodiment, the inventionrelates to a method for producing a nuclear receptor ligand bindingdomain comprising a substitution mutation, wherein the method comprisesculturing the host cell as described above in culture medium underconditions permitting expression of a polynucleotide encoding thenuclear receptor ligand binding domain comprising a substitutionmutation, and isolating the nuclear receptor ligand binding domaincomprising a substitution mutation from the culture.

In a specific embodiment, the isolated host cell is a prokaryotic hostcell or a eukaryotic host cell. In another specific embodiment, theisolated host cell is an invertebrate host cell or a vertebrate hostcell. Preferably, the host cell is selected from the group consisting ofa bacterial cell, a fungal cell, a yeast cell, a nematode cell, aninsect cell, a fish cell, a plant cell, an avian cell, an animal cell,and a mammalian cell. More preferably, the host cell is a yeast cell, anematode cell, an insect cell, a plant cell, a zebrafish cell, a chickencell, a hamster cell, a mouse cell, a rat cell, a rabbit cell, a catcell, a dog cell, a bovine cell, a goat cell, a cow cell, a pig cell, ahorse cell, a sheep cell, a simian cell, a monkey cell, a chimpanzeecell, or a human cell. Examples of preferred host cells include, but arenot limited to, fungal or yeast species such as Aspergillus,Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterialspecies such as those in the genera Synechocystis, Synechococcus,Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces,Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes,Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella;plant species selected from the group consisting of an apple,Arabidopsis, bajra, banana, barley, beans, beet, blackgram, chickpea,chili, cucumber, eggplant, favabean, maize, melon, millet, mungbean,oat, okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple,Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash, sugarcane,sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon,and wheat; animal; and mammalian host cells.

In a specific embodiment, the host cell is a yeast cell selected fromthe group consisting of a Saccharomyces, a Pichia, and a Candida hostcell.

In another specific embodiment, the host cell is a Caenorhabdus elegansnematode cell.

In another specific embodiment, the host cell is an insect cell.

In another specific embodiment, the host cell is a plant cell selectedfrom the group consisting of an apple, Arabidopsis, bajra, banana,barley, beans, beet, blackgram, chickpea, chili, cucumber, eggplant,favabean, maize, melon, millet, mungbean, oat, okra, Panicum, papaya,peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin,rice, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweetpotato, tea, tomato, tobacco, watermelon, and wheat cell.

In another specific embodiment, the host cell is a zebrafish cell.

In another specific embodiment, the host cell is a chicken cell.

In another specific embodiment, the host cell is a mammalian cellselected from the group consisting of a hamster cell, a mouse cell, arat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goatcell, a cow cell, a pig cell, a horse cell, a sheep cell, a monkey cell,a chimpanzee cell, and a human cell.

Host cell transformation is well known in the art and may be achieved bya variety of methods including but not limited to electroporation, viralinfection, plasmid/vector transfection, non-viral vector mediatedtransfection, Agrobacterium-mediated transformation, particlebombardment, and the like. Expression of desired gene products involvesculturing the transformed host cells under suitable conditions andinducing expression of the transformed gene. Culture conditions and geneexpression protocols in prokaryotic and eukaryotic cells are well knownin the art (see General Methods section of Examples). Cells may beharvested and the gene products isolated according to protocols specificfor the gene product.

In addition, a host cell may be chosen which modulates the expression ofthe inserted polynucleotide, or modifies and processes the polypeptideproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification [e.g., glycosylation,cleavage (e.g., of signal sequence)] of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce a non-glycosylated coreprotein product. However, a polypeptide expressed in bacteria may not beproperly folded. Expression in yeast can produce a glycosylated product.Expression in eukaryotic cells can increase the likelihood of “native”glycosylation and folding of a heterologous protein. Moreover,expression in mammalian cells can provide a tool for reconstituting, orconstituting, the polypeptide's activity. Furthermore, differentvector/host expression systems may affect processing reactions, such asproteolytic cleavages, to a different extent.

Applicants' invention also relates to a non-human organism comprising anisolated host cell according to the invention. In a specific embodiment,the non-human organism is a prokaryotic organism or a eukaryoticorganism. In another specific embodiment, the non-human organism is aninvertebrate organism or a vertebrate organism.

Preferably, the non-human organism is selected from the group consistingof a bacterium, a fungus, a yeast, a nematode, an insect, a fish, aplant, a bird, an animal, and a mammal. More preferably, the non-humanorganism is a yeast, a nematode, an insect, a plant, a zebrafish, achicken, a hamster, a mouse, a rat, a rabbit, a cat, a dog, a bovine, agoat, a cow, a pig, a horse, a sheep, a simian, a monkey, or achimpanzee.

In a specific embodiment, the non-human organism is a yeast selectedfrom the group consisting of Saccharomyces, Pichia, and Candida.

In another specific embodiment, the non-human organism is a Caenorhabduselegans nematode.

In another specific embodiment, the non-human organism is a plantselected from the group consisting of an apple, Arabidopsis, bajra,banana, barley, beans, beet, blackgram, chickpea, chili, cucumber,eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum,papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato,pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet,sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat.

In another specific embodiment, the non-human organism is a Mus musculusmouse.

Measuring Gene Expression/Transcription

One useful measurement of Applicants' methods of the invention is thatof the transcriptional state of the cell including the identities andabundances of RNA, preferably mRNA species. Such measurements areconveniently conducted by measuring cDNA abundances by any of severalexisting gene expression technologies.

Nucleic acid array technology is a useful technique for determiningdifferential mRNA expression. Such technology includes, for example,oligonucleotide chips and DNA microarrays. These techniques rely on DNAfragments or oligonucleotides which correspond to different genes orcDNAs which are immobilized on a solid support and hybridized to probesprepared from total mRNA pools extracted from cells, tissues, or wholeorganisms and converted to cDNA. Oligonucleotide chips are arrays ofoligonucleotides synthesized on a substrate using photolithographictechniques. Chips have been produced which can analyze for up to 1700genes. DNA microarrays are arrays of DNA samples, typically PCRproducts, that are robotically printed onto a microscope slide. Eachgene is analyzed by a full or partial-length target DNA sequence.Microarrays with up to 10,000 genes are now routinely preparedcommercially. The primary difference between these two techniques isthat oligonucleotide chips typically utilize 25-mer oligonucleotideswhich allow fractionation of short DNA molecules whereas the larger DNAtargets of microarrays, approximately 1000 base pairs, may provide moresensitivity in fractionating complex DNA mixtures.

Another useful measurement of Applicants' methods of the invention isthat of determining the translation state of the cell by measuring theabundances of the constituent protein species present in the cell usingprocesses well known in the art.

Where identification of genes associated with various physiologicalfunctions is desired, an assay may be employed in which changes in suchfunctions as cell growth, apoptosis, senescence, differentiation,adhesion, binding to a specific molecules, binding to another cell,cellular organization, organogenesis, intracellular transport, transportfacilitation, energy conversion, metabolism, myogenesis, neurogenesis,and/or hematopoiesis is measured.

In addition, selectable marker or reporter gene expression may be usedto measure gene expression modulation using Applicants' invention.

Other methods to detect the products of gene expression are well knownin the art and include Southern blots (DNA detection), dot or slot blots(DNA, RNA), northern blots (RNA), RT-PCR (RNA), western blots(polypeptide detection), and ELISA (polypeptide) analyses. Although lesspreferred, labeled proteins can be used to detect a particular nucleicacid sequence to which it hybidizes.

In some cases it is necessary to amplify the amount of a nucleic acidsequence. This may be carried out using one or more of a number ofsuitable methods including, for example, polymerase chain reaction(“PCR”), ligase chain reaction (“LCR”), strand displacementamplification (“SDA”), transcription-based amplification, and the like.PCR is carried out in accordance with known techniques in which, forexample, a nucleic acid sample is treated in the presence of a heatstable DNA polymerase, under hybridizing conditions, with one pair ofoligonucleotide primers, with one primer hybridizing to one strand(template) of the specific sequence to be detected. The primers aresufficiently complementary to each template strand of the specificsequence to hybridize therewith. An extension product of each primer issynthesized and is complementary to the nucleic acid template strand towhich it hybridized. The extension product synthesized from each primercan also serve as a template for further synthesis of extension productsusing the same primers. Following a sufficient number of rounds ofsynthesis of extension products, the sample may be analyzed as describedabove to assess whether the sequence or sequences to be detected arepresent.

Ligand Screening Assays

The present invention also relates to methods of screening for acompound that induces or represses transactivation of a nuclear receptorligand binding domain comprising a substitution mutation in a cell bycontacting a nuclear receptor ligand binding domain with a candidatemolecule and detecting reporter gene activity in the presence of theligand. Candidate compounds may be either agonists or antagonists of thenuclear receptor ligand binding domain. In a preferred embodiment, thenuclear receptor ligand binding domain is expressed from apolynucleotide in the cell and the transactivation activity (i.e.,expression or repression of a reporter gene) or compound bindingactivity is measured.

Accordingly, in addition to rational design of agonists and antagonistsbased on the structure of a nuclear receptor ligand binding domain, thepresent invention contemplates an alternative method for identifyingspecific ligands of a nuclear receptor ligand binding domain usingvarious screening assays known in the art.

Any screening technique known in the art can be used to screen for GroupH nuclear receptor ligand binding domain agonists or antagonists. Forexample, a suitable cell line comprising a nuclear receptor-based geneexpression system according to the invention can be transfected with agene expression cassette encoding a marker gene operatively linked to aninducible or repressible promoter. The transfected cells are thenexposed to a test solution comprising a candidate agonist or antagonistcompound, and then assayed for marker gene expression or repression. Thepresence of more marker gene expression relative to control cells notexposed to the test solution is an indication of the presence of anagonist compound in the test solution. Conversely, the presence of lessmarker gene expression relative to control cells not exposed to the testsolution is an indication of the presence of an antagonist compound inthe test solution.

The present invention contemplates screens for small molecule ligands orligand analogs and mimics, as well as screens for natural ligands thatbind to and agonize or antagonize a Group H nuclear receptor ligandbinding domain according to the invention in vivo. For example, naturalproducts libraries can be screened using assays of the invention formolecules that agonize or antagonize nuclear receptor-based geneexpression system activity.

Identification and screening of antagonists is further facilitated bydetermining structural features of the protein, e.g., using X-raycrystallography, neutron diffraction, nuclear magnetic resonancespectrometry, and other techniques for structure determination. Thesetechniques provide for the rational design or identification of agonistsand antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” [Scott and Smith, 1990, Science 249:386-390 (1990); Cwirla, et al., Proc. Natl. Acad. Sci., 87: 6378-6382(1990); Devlin et al., Science, 249: 404-406 (1990)], very largelibraries can be constructed (10⁶-10⁸ chemical entities). A secondapproach uses primarily chemical methods, of which the Geysen method[Geysen et al., Molecular Immunology 23: 709-715 (1986); Geysen et al.J. Immunologic Method 102: 259-274 (1987)] and the method of Fodor etal. [Science 251: 767-773 (1991)] are examples. Furka et al. [14thInternational Congress of Biochemistry, Volume 5, Abstract FR:013(1988); Furka, Int. J. Peptide Protein Res. 37:487-493 (1991)], Houghton[U.S. Pat. No. 4,631,211, issued December 1986] and Rutter et al. [U.S.Pat. No. 5,010,175, issued Apr. 23, 1991] describe methods to produce amixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries [Needels et al., Proc. Natl.Acad. Sci. USA 90: 10700-4 (1993); Ohlmeyer et al., Proc. Natl. Acad.Sci. USA 90: 10922-10926 (1993); Lam et al., International PatentPublication No. WO 92/00252; Kocis et al., International PatentPublication No. WO 9428028, each of which is incorporated herein byreference in its entirety], and the like can be used to screen forcandidate ligands according to the present invention.

The screening can be performed with recombinant cells that express anuclear receptor ligand binding domain according to the invention, oralternatively, using purified protein, e.g., produced recombinantly, asdescribed above. For example, labeled, soluble nuclear receptor ligandbinding domains can be used to screen libraries, as described in theforegoing references.

In one embodiment, a Group H nuclear receptor ligand binding domainaccording to the invention may be directly labeled. In anotherembodiment, a labeled secondary reagent may be used to detect binding ofa nuclear receptor ligand binding domain of the invention to a moleculeof interest, e.g., a molecule attached to a solid phase support. Bindingmay be detected by in situ formation of a chromophore by an enzymelabel. Suitable enzymes include, but are not limited to, alkalinephosphatase and horseradish peroxidase. In a further embodiment, atwo-color assay, using two chromogenic substrates with two enzyme labelson different acceptor molecules of interest, may be used. Cross-reactiveand singly-reactive ligands may be identified with a two-color assay.

Other labels for use in the invention include colored latex beads,magnetic beads, fluorescent labels (e.g., fluorescene isothiocyanate(FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelatedlanthanide series salts, especially Eu³⁺, to name a few fluorophores),chemiluminescent molecules, radio-isotopes, or magnetic resonanceimaging labels. Two-color assays may be performed with two or morecolored latex beads, or fluorophores that emit at different wavelengths.Labeled molecules or cells may be detected visually or bymechanical/optical means. Mechanical/optical means include fluorescenceactivated sorting, i.e., analogous to FACS, and micromanipulator removalmeans.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention.

EXAMPLES

Applicants have developed a CfEcR homology model and have used thishomology model together with a published Chironomous tetans ecdysonereceptor (“CtEcR”) homology model (Wurtz et al., 2000) to identifycritical residues involved in binding to steroids and non-steroids. Thesynthetic non-steroids, diacylhydrazines, have been shown to bindlepidopteran EcRs with high affinity and induce precocious incompletemolt in these insects (Wing et al., 1988) and several of these compoundsare currently marketed as insecticides. The ligand binding cavity ofEcRs has evolved to fit the long back bone structures of ecdysteroidssuch as 20E. The diacylhydrazines have a compact structure compared tosteroids and occupy only the bottom part of the EcR binding pocket. Thisleaves a few critical residues at the top part of the binding pocketthat make contact with steroids but not with non-steroids such asdiacylhydrazines. Applicants made substitution mutations of the residuesthat make contact with steroids and/or non-steroids and determined themutational effect on ligand binding. Applicants describe hereinsubstitution mutations at several of these residues and have identifiedseveral classes of substitution mutant receptors based upon theirbinding and transactivation characteristics. Applicants' novelsubstitution mutated nuclear receptor polynucleotides and polypeptidesare useful in a nuclear receptor-based inducible gene modulation systemfor various applications including gene therapy, expression of proteinsof interest in host cells, production of transgenic organisms, andcell-based assays.

General Methods

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) (Maniatis) andby T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984)and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials used for the growth and maintenance of host cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma ChemicalCompany (St. Louis, Mo.) unless otherwise specified.

Manipulations of genetic sequences may be accomplished using the suiteof programs available from the Genetics Computer Group Inc. (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.).Where the GCG program “Pileup” is used the gap creation default value of12, and the gap extension default value of 4 may be used. Where the CGC“Gap” or “Bestfit” program is used the default gap creation penalty of50 and the default gap extension penalty of 3 may be used. In any casewhere GCG program parameters are not prompted for, in these or any otherGCG program, default values may be used.

The meaning of abbreviations is as follows: “h” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “μl” meansmicroliter(s), “ml” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “μg” means microgram(s), “mg” meansmilligram(s), “A” means adenine or adenosine, “T” means thymine orthymidine, “G” means guanine or guanosine, “C” means cytidine orcytosine, “xg” means times gravity, “nt” means nucleotide(s), “aa” meansamino acid(s), “bp” means base pair(s), “kb” means kilobase(s), “k”means kilo, “μ” means micro, and “° C.” means degrees Celsius.

Example 1

This Example describes the construction of several gene expressioncassettes comprising novel substitution mutated Group H nuclear receptorpolynucleotides and polypeptides of the invention for use in a nuclearreceptor-based inducible gene expression system. Applicants constructedseveral gene expression cassettes based on the spruce budwormChoristoneura fumiferana EcR (“CfEcR”), fruit fly Drosophilamelanogaster EcR (“DmEcR”), ixodid tick Amblyomma americanum EcR(“AmaEcR”), locust Locusta migratoria ultraspiracle protein (“LmUSP”),an invertebrate RXR homolog of vertebrate RXR, and C. fumiferana USP(“CfUSP”). The prepared receptor constructs comprise a ligand bindingdomain of either an EcR, an invertebrate USP, or an invertebrate RXR;and a GAL4 DNA binding domain (DBD) or a VP16 transactivation domain(AD). The reporter constructs include a reporter gene, luciferase orLacZ (β-galactosidase), operably linked to a synthetic promoterconstruct that comprises a GAL4 response element to which the Gal4 DBDbinds. Various combinations of these receptor and reporter constructswere cotransfected into mammalian cells as described in Examples 2-10infra.

Gene Expression Cassettes:

Ecdysone receptor-based gene expression cassettes (switches) wereconstructed as followed, using standard cloning methods available in theart. The following is a brief description of preparation and compositionof each switch used in the Examples described herein.

1.1—GAL4CfEcR-DEF/VP16LmUSP-EF:

The wild-type D, E, and F domains from spruce budworm Choristoneurafumiferana EcR (“CfEcR-DEF”; SEQ ID NO: 21) were fused to a GAL4 DNAbinding domain (“Gal4DNABD” or “Gal4 DBD”; SEQ ID NO: 6) and placedunder the control of an SV40e promoter (SEQ ID NO: 22). The E and Fdomains from locust Locusta migratoria ultraspiracle protein(“LmUSP-EF”; SEQ ID NO: 23) were fused to the transactivation domainfrom VP16 (“VP16AD”; SEQ ID NO: 12) and placed under the control of anSV40e promoter (SEQ ID NO: 22). Five consensus GAL4 response elementbinding sites (“5XGAL4RE”; comprising 5 copies of a GAL4RE comprisingSEQ ID NO: 19) were fused to a synthetic E1b minimal promoter (SEQ IDNO: 24) and placed upstream of the luciferase gene (SEQ ID NO: 25).

1.2—GAL4/mutantCfEcR-DEF/VP16LmUSP-EF:

This construct was prepared in the same way as in switch 1.1 aboveexcept wild-type CfEcR-DEF was replaced with mutant CfEcR-DEF comprisinga ligand binding domain comprising a substitution mutation selected fromTable 1 below.

TABLE 1 Substitution Mutants of Choristoneura fumiferana EcdysoneReceptor (“CfEcR”) Ligand Binding Domain (LBD). Corresponding amino acidin CfEcR-DEF Resulting “WT to Mutant” Amino full length CfEcR LBDMutation Acid Substitution (SEQ ID NO: 26) E20A Glutamic Acid (E) toAlanine (A) 303 Q21A Glutamine (Q) to Alanine (A) 304 F48A Phenylalanine(F) to Alanine (A) 331 I51A Isoleucine (I) to Alanine (A) 334 T52AThreonine (T) to Alanine (A) 335 T52L Threonine (T) to Leucine (L) 335T52V Threonine (T) to Valine (V) 335 T52I Threonine (T) to Isoleucine(I) 335 T55A Threonine (T) to Alanine (A) 338 T58A Threonine (T) toAlanine (A) 341 V59A Valine (V) to Alanine (A) 342 L61A Leucine (L) toAlanine (A) 344 I62A Isoleucine (I) to Alanine (A) 345 M92A Methionine(M) to Alanine (A) 375 M93A Methionine (M) to Alanine (A) 376 R95AArginine (R) to Alanine (A) 378 V96A Valine (V) to Alanine (A) 379 V96TValine (V) to Threonine (T) 379 V96D Valine (V) to Aspartic Acid (D) 379V96M Valine (V) to Methionine (M) 379 V107I Valine (V) to Isoleucine 390F109A Phenylalanine (F) to Alanine (A) 392 A110P Alanine (A) to Proline(P) 393 A110S Alanine (A) to Serine (S) 393 A110L Alanine (A) to Leucine(L) 393 A110M Alanine (A) to Methionine (M) 393 Y120A Tyrosine (Y) toAlanine (A) 403 A123F Alanine (A) to Phenylalanine (F) 406 M125AMethionine (M) to Alanine (A) 408 R175E Arginine (R) to Glutamine (E)458 M218A Methionine (M) to Alanine (A) 501 C219A Cysteine (C) toAlanine (A) 502 L223A Leucine (L) to Alanine (A) 506 L230A Leucine (L)to Alanine (A) 513 L234A Leucine (L) to Alanine (A) 517 W238A Tryptophan(W) to Alanine (A) 521 R95A and Arginine (R) to Alanine (A) and 378 and393, A110P double Alanine (A) to Proline (P), respectively mutantrespectively V107I and Valine (V) to Isoleucine (I) and 390 and 458,R175E double Arginine (R) to Glutamine (E), respectively mutantrespectively V107E and Valine (V) to Isoleucine (I) and 390 and 410,Y127E double Tyrosine (Y) to Glutamine (E), respectively mutantrespectively Y127E and Tyrosine (Y) to Glutamine (E) and 410 and 458,R175E double Arginine (R) to Glutamine (E), respectively mutantrespectively M218A and Methionine (M) to Alanine (A) and 501 and 502,C219A double Cysteine (C) to Alanine (A), respectively mutantrespectively T52V, V107I Threonine (T) to Valine (V), 335, 390 and 458,and R175E Valine (V) to Isoleucine (I) and respectively triple Arginine(R) to Glutamine (E), mutant respectively T52A, V107I Threonine (T) toAlanine (A), 335, 390 and 458, and R175E Valine (V) to Isoleucine (I)and respectively triple Arginine (R) to Glutamine (E), mutantrespectively V96A, V107I Valine (V) to Alanine (A), 379, 390 and 458,and R175E Valine (V) to Isoleucine (I) and respectively triple Arginine(R) to Glutamine (E), mutant respectively V96T, V107I Valine (V) toThreonine (T), 379, 390 and 458, and R175E Valine (V) to Isoleucine (I)and respectively triple Arginine (R) to Glutamine (E), mutantrespectively V107I, Y127E Valine (V) to Isoleucine (I), 390, 410 and458, and R175E Tyrosine (Y) to Glutamine (E) and respectively tripleArginine (R) to Glutamine (E), mutant respectively V107I, A110P Valine(V) to Isoleucine (I), 390, 393, and 458, and R175E Alanine (A) toProline (P) and respectively triple Arginine (R) to Glutamine (E),mutant respectively

1.3—GAL4CfEcR-A/BCDEF/VP16LmUSP-EF:

The full-length spruce budworm Choristoneura fumiferana EcR(“CfEcR-A/BCDEF”; SEQ ID NO: 27) was fused to a GAL4 DNA binding domain(“Gal4DNABD” or “Gal4 DBD”; SEQ ID NO: 6) and placed under the controlof an SV40e promoter (SEQ ID NO: 22). The E and F domains from Locustamigratoria ultraspiracle (“LmUSP-EF”; SEQ ID NO: 23) were fused to thetransactivation domain from VP16 (“VP16AD”; SEQ ID NO: 12) and placedunder the control of an SV40e promoter (SEQ ID NO: 22). Five consensusGAL4 response element binding sites (“5XGAL4RE”; comprising 5 copies ofa GAL4RE comprising SEQ ID NO: 19) were fused to a synthetic E1b minimalpromoter (SEQ ID NO: 24) and placed upstream of the luciferase gene (SEQID NO: 25).

1.4—GAL4/A110PmutantCfEcR-A/BCDEF/VP16LmUSP-EF:

This construct was prepared in the same way as in switch 1.3 aboveexcept wild-type CfEcR-A/BCDEF was replaced with a mutant CfEcR-A/BCDEFcomprising a ligand binding domain comprising an A110P substitutionmutation as described in Table 1 above.

1.5—VP16/CfEcR-CDEF:

This construct was prepared in the same way as switch 1.1 except theGAL4 DNA binding domain was replaced with the transactivation domainfrom VP16 (“VP16AD”; SEQ ID NO: 12) and placed under the control of abaculovirus IE1 promoter (SEQ ID NO: 28). Six consensus ecdysoneresponse element binding sites (“6XEcRE”; comprising 6 copies of anecdysone RE comprising SEQ ID NO: 18) were fused to a synthetic E1bminimal promoter (SEQ ID NO: 24) and placed upstream of theβ-galactosidase gene (SEQ ID NO: 29). This construct uses endogenousultraspiracle protein as a heterodimerization partner.

1.6—VP16/A110PmutantCfEcR-CDEF:

This construct was prepared in the same way as in switch 1.5 aboveexcept wild-type CfEcR-CDEF was replaced with a mutant CfEcR-CDEFcomprising a ligand binding domain comprising an A110P substitutionmutation as described in Table 1 above.

1.7—Bacterially expressed CfUSP-A/BCDEF:

This construct was prepared with the A/BCDEF domains from spruce budwormC. fumiferana USP (“CfUSP-A/BCDEF”; SEQ ID NO: 30).

1.8—GAL4/DmEcR-CDEF/VP16LmUSP-EF:

The wild-type C, D, E, and F domains from fruit fly Drosophilamelanogaster EcR (“DmEcR-CDEF”; SEQ ID NO: 31) were fused to a GAL4 DNAbinding domain (“Gal4DNABD” or “Gal4 DBD”; SEQ ID NO: 6) and placedunder the control of an SV40e promoter (SEQ ID NO: 22). The E and Fdomains from locust Locusta migratoria ultraspiracle protein(“LmUSP-EF”; SEQ ID NO: 23) were fused to the transactivation domainfrom VP16 (“VP16AD”; SEQ ID NO: 12) and placed under the control of anSV40e promoter (SEQ ID NO: 22). Five consensus GAL4 response elementbinding sites (“5XGAL4RE”; comprising 5 copies of a GAL4RE comprisingSEQ ID NO: 19) were fused to a synthetic E1b minimal promoter (SEQ IDNO: 24) and placed upstream of the luciferase gene (SEQ ID NO: 25).

1.9—GAL4/mutantDmEcR-CDEF/VP16LmUSP-EF:

This construct was prepared in the same way as in switch 1.8 aboveexcept wild-type DmEcR-CDEF was replaced with mutant DmEcR-CDEFcomprising a ligand binding domain comprising a substitution mutationselected from Table 2 below.

TABLE 2 Substitution Mutants of Drosophila melanogaster EcdysoneReceptor (“DmEcR”) Ligand Binding Domain (LBD). Corresponding amino acidin DmEcR-CDEF Resulting “WT to Mutant” Amino full length DmEcR LBDMutation Acid Substitution (SEQ ID NO: 32) A107P Alanine (A) to Proline(P) 522 G121R Glycine (G) to Arginine (R) 536 G121L Glycine (G) toLeucine (L) 536 N213A Asparagine (N) to Alanine (A) 628 C217A Cysteine(C) to Alanine (A) 632 C217S Cysteine (C) to Serine (S) 632

1.10—VP16/DmEcR-CDEF:

This construct was prepared in the same way as switch 1.8 except theGAL4 DNA binding domain was replaced with the transactivation domainfrom VP16 (“VP16AD”; SEQ ID NO: 12) and placed under the control of abaculovirus IE1 promoter (SEQ ID NO: 28). Six consensus ecdysoneresponse element binding sites (“6XEcRE”; comprising 6 copies of anecdysone RE comprising SEQ ID NO: 18) were fused to a synthetic E1bminimal promoter (SEQ ID NO: 24) and placed upstream of theβ-galactosidase gene (SEQ ID NO: 29). This construct uses endogenousultraspiracle protein as a heterodimerization partner.

1.11—VP16/mutantDmEcR-CDEF:

This construct was prepared in the same way as in switch 1.10 aboveexcept wild-type DmEcR-CDEF was replaced with a mutant DmEcR-CDEFcomprising a ligand binding domain comprising a substitution mutationselected from Table 2 above.

1.12—GAL4/AmaEcR-DEF/VP16LmUSP-EF:

The wild-type D, E, and F domains from ixodid tick Amblyomma americanumEcR (“AmaEcR-DEF”; SEQ ID NO: 33) were fused to a GAL4 DNA bindingdomain (“Gal4DNABD” or “Gal4 DBD”; SEQ ID NO: 6) and placed under thecontrol of an SV40e promoter (SEQ ID NO: 22). The E and F domains fromlocust Locusta migratoria ultraspiracle protein (“LmUSP-EF”; SEQ ID NO:23) were fused to the transactivation domain from VP16 (“VP16AD”; SEQ IDNO: 12) and placed under the control of an SV40e promoter (SEQ ID NO:22). Five consensus GAL4 response element binding sites (“5XGAL4RE”;comprising 5 copies of a GAL4RE comprising SEQ ID NO: 19) were fused toa synthetic E1b minimal promoter (SEQ ID NO: 24) and placed upstream ofthe luciferase gene (SEQ ID NO: 25).

1.13—GAL4/mutantAmaEcR-DEF/VP16LmUSP-EF:

This construct was prepared in the same way as in switch 1.12 aboveexcept wild-type AmaEcR-DEF was replaced with mutant AmaEcR-DEFcomprising a ligand binding domain comprising a substitution mutationselected from Table 3 below.

TABLE 3 Substitution Mutants of Amblyomma americanum Ecdysone Receptor(“AmaEcR”) Ligand Binding Domain (LBD). Corresponding amino acid inAmaEcR-DEF Resulting “WT to Mutant” Amino full length AmaEcR LBDMutation Acid Substitution (SEQ ID NO: 34) G91A Glycine (G) to Alanine(A) 417 A105P Alanine (A) to Proline (P) 431

Construction of Ecdysone Receptor Ligand Binding Domains Comprising aSubstitution Mutation:

In an effort to modify EcR ligand binding, residues within the EcRligand binding domains that were predicted to be important for ligandbinding based upon a molecular modeling analysis were mutated in EcRsfrom three different classes of organisms. Tables 1-3 indicate the aminoacid residues within the ligand binding domain of CfEcR (LepidopteranEcR), DmEcR (Dipteran EcR) and AmaEcR (Arthopod EcR), respectively thatwere mutated and examined for modification of steroid and non-steroidbinding.

Each one of the amino acid substitution mutations listed in Tables 1-3was constructed in an EcR cDNA by PCR mediated site-directed mutagenesisto alanine (or to proline or phenylalanine in the case of a wild-typealanine residue, e.g. CfEcR residues A110 and A123, respectively). Aminoacids T52, V96 and A110 of CfEcR were mutated to four different aminoacids. Five different double point mutant CfEcRs were also made: onecomprising both the R95A and A110P substitutions (R95A+A110P, “DM”), asecond comprising both the M218A and C219A substitutions (M218A+C219A),a third comprising both the V107I and R175E substitutions (V107I+R175E),a fourth comprising Y127E and R175E substitutions (Y127E+R175E), and afifth comprising V107I and Y127E substitutions (V107I+Y127E). Sixdifferent triple point mutant CfEcRs were also made: one comprising boththe V107I and R175E substitutions and a Y127E substitution(V107I+Y127E+R175E), a second comprising a T52V substitution and theV107I and R175E substitutions (T52V+V107I+R175E), a third comprising theV96A, V107I, and R175E substitutions (V96A+V107I+R175E), a fourthcomprising the T52A, V107I and R175E substitutions (T52A+V107I+R175E), afifth comprising a V96T substitution and the V107I and R175Esubstitutions (V96T+V107I+R175E), and a sixth comprising the A110P,V107I, and R175E substitutions.

PCR site-directed mutagenesis was performed using the Quikchangesite-directed mutagenesis kit (Stratagene, La Jolla, Calif.) using thereaction conditions and cycling parameters as follows. PCR site-directedmutagenesis was performed using 1× reaction buffer (supplied bymanufacturer), 50 ng of dsDNA template, 125 ng of forward primer (FP),125 ng of reverse complementary primer (RCP), and 1 μl of dNTP mix(supplied by manufacturer) in a final reaction volume of 50 μL. Theforward primer and reverse complementary primer used to produce each EcRmutant are presented in Tables 4-6. The cycling parameters usedconsisted of one cycle of denaturing at 95° C. for 30 seconds, followedby 16 cycles of denaturating at 95° C. for 30 seconds, annealing at 55°C. for 1 minute, and extending at 68° C. for 22 minutes.

TABLE 4 PCR Primers for Substitution Mutant CfEcR Ligand Binding Domain ConstructionPRIMER MUTANT (SEQ ID NO:) PRIMER NUCLEOTIDE SEQUENCE (5′ TO 3′) E20A FPgtaccaggacgggtacgcgcagccttctgatgaagatttg (SEQ ID NO: 35) E20A RCPcaaatcttcatcagaaggctgcgcgtacccgtcctggtac (SEQ ID NO: 36) Q21A FPccaggacgggtacgaggcgccttctgatgaagatttg (SEQ ID NO: 37) Q21A RCPcaaatcttcatcagaaggcgcctcgtacccgtcctgg (SEQ ID NO: 38) F48A FPgagtctgacactcccgcccgccagatcacag (SEQ ID NO: 39) F48A RCPctgtgatctggcgggcgggagtgtcagactc (SEQ ID NO: 40) I51A FPcactcccttccgccaggccacagagatgac (SEQ ID NO: 41) I51A RCPgtcatctctgtggcctggcggaagggagtg (SEQ ID NO: 42) T52A FPcactcccttccgccagatcgcagagatgac (SEQ ID NO: 43) T52A RCPgtcatctctgcgatctggcggaagggagtg (SEQ ID NO: 44) T55A FPcgccagatcacagagatggctatcctcacggtcc (SEQ ID NO: 45) T55A RCPggaccgtgaggatagccatctctgtgatctggcg (SEQ ID NO: 46) T58A FPgagatgactatcctcgcggtccaacttatcgtg (SEQ ID NO: 47) T58A RCPcacgataagttggaccgcgaggatagtcatctc (SEQ ID NO: 48) V59A FPgatgactatcctcacggcccaacttatcgtgg (SEQ ID NO: 49) V59A RCPccacgataagttgggccgtgaggatagtcatc (SEQ ID NO: 50) L61A FPctatcctcacggtccaagctatcgtggagttcgcg (SEQ ID NO: 51) L61A RCPcgcgaactccacgatagcttggaccgtgaggatag (SEQ ID NO: 52) I62A FPctatcctcacggtccaacttgccgtggagttcgcg (SEQ ID NO: 53) 162A RCPcgcgaactccacggcaagttggaccgtgaggatag (SEQ ID NO: 54) M92A FPgctcaagtgaggtagcgatgctccgagtcgc (SEQ ID NO: 55) M92A RCPgcgactcggagcatcgctacctcacttgagc (SEQ ID NO: 56) M93A FPgctcaagtgaggtaatggcgctccgagtcgc (SEQ ID NO: 57) M93A RCPgcgactcggagcgccattacctcacttgagc (SEQ ID NO: 58) V96A FPgtaatgatgctccgagccgcgcgacgatac (SEQ ID NO: 59) V96A RCPgtatcgtcgcgcggctcggagcatcattac (SEQ ID NO: 60) R95A FPgtgaggtaatgatgctcgcagtcgcgcgacgatacg (SEQ ID NO: 61) R95A RCPcgtatcgtcgcgcgactgcgagcatcattacctcac (SEQ ID NO: 62) F108A FPcagacagtgttctggccgcgaacaaccaagcg (SEQ ID NO: 63) F108A RCPcgcttggttgttcgcggccagaacactgtctg (SEQ ID NO: 64) F109A FPtcagacagtgttctggccgcgaacaaccaagcg (SEQ ID NO: 65) F109A RCPcgcttggttgttcgcggccagaacactgtctga (SEQ ID NO: 66) A110P FPcagacatgttctgttcccgaacaaccaagcg (SEQ ID NO: 67) A110P RCPcgcttggttgttcgggaacagaacactgtctg (SEQ ID NO: 68) Y120A FPcactcgcgacaacgcccgcaaggctggcatg (SEQ ID NO: 69) Y120A RCPcatgccagccttgcgggcgttgtcgcgagtg (SEQ ID NO: 70) A123F FPcgacaactaccgcaagtttggcatggcctacgtc (SEQ ID NO: 71) A123F RCPgacgtaggccatgccaaacttgcggtagttgtcg (SEQ ID NO: 72) M125A FPctaccgcaaggctggcgcggcctacgtcatc (SEQ ID NO: 73) M125A RCPgatgacgtaggccgcgccagccttgcggtag (SEQ ID NO: 74) L230A FPgctcaagaacagaaaggcgccgcctttcctcg (SEQ ID NO: 75) L230A RCPcgaggaaaggcggcgcctttctgttcttgagc (SEQ ID NO: 76) L223A FPctccaacatgtgcatctccgccaagctcaagaacag (SEQ ID NO: 77) L223A RCPctgttcttgagcttggcggagatgcacatgttggag (SEQ ID NO: 78) L234A FPgaaagctgccgcctttcgccgaggagatctg (SEQ ID NO: 79) L234A RCPcagatctcctcggcgaaaggcggcagctttc (SEQ ID NO: 80) W238A FPctttcctcgaggagatcgcggatgtggcagg (SEQ ID NO: 81) W238A RCPcctgccacatccgcgatctcctcgaggaaag (SEQ ID NO: 82) A110n RANDOM FPcagacagtgttctgttgncgaacaaccaagcg (SEQ ID NO: 83) A110n RANDOM RCPcgcttggttgttcgncaacagaacactgtctg (SEQ ID NO: 84) A110n RANDOM2 FP cagacagtgttctgttgnngaacaaccaagcg (SEQ ID NO: 85) A110n RANDOM2 RCPcgcttggttgttcnncaacagaacactgtctg (SEQ ID NO: 86) T52n RANDOM FPcactcccttccgccagatcnnngagatgactatcctcacg (SEQ ID NO: 87) T52n RANDOM RCPcgtgaggatagtcatctcnnngatctggcggaagggagt (SEQ ID NO: 88) V96n RANDOM FPgtaatgatgctccgannngcgcgacgatacgatgcggc (SEQ ID NO: 89) V96n RANDOM RCPgccgcatcgtatcgtcgcgcnnntcggagcatcattac (SEQ ID NO: 90) V107I FPgcggcctcagacagtattctgttcgcgaac (SEQ ID NO: 107) R175E FPggtggaagaaatccaggagtactacctgaatacgctcc (SEQ ID NO: 108) Y127E FPcaaggctggcatggccgaggtcatcgagg (SEQ ID NO: 109) T52V FPcccttccgccagatcgtagagatgactatcctcac (SEQ ID NO: 110) V96T FPggtaatgatgctccgaaccgcgcgacgatacg (SEQ ID NO: 111) 

TABLE 5 PCR Primers for Substitution Mutant DmEcR Ligand Binding Domain ConstructionPRIMER MUTANT (SEQ ID NO:) PRIMER NUCLEOTIDE SEQUENCE (5′ TO 3′) A107PFP tcggactcaatattcttcccgaataatagatcatatac (SEQ ID NO: 91) A107P RCPgtatatgatctattattcgggaagaatattgagtccga (SEQ ID NO: 92) G121R FPtcttacaaaatggcccgaatggctgataacattg (SEQ ID NO: 93) G121R RCPcaatgttatcagccattcgggccattttgtaaga (SEQ ID NO: 94) G121L FPtcttacaaaatggccctaatggctgataacattg (SEQ ID NO: 95) G121L RCPcaatgttatcagccattagggccattttgtaaga (SEQ ID NO: 96) N213A FPacgctgggcaaccaggccgccgagatgtgtttc (SEQ ID NO: 97) N213A RCPgaaacacatctcggcggcctggttgcccagcgt (SEQ ID NO: 98) C217A FPcagaacgccgagatggctttctcactaaagctc (SEQ ID NO: 99) C217A RCPgagctttagtgagaaagccatcteggcgttctg (SEQ ID NO: 100) C217S FPcagaacgccgagatgtctttctcactaaagctc (SEQ ID NO: 101) C217S RCPgagctttagtgagaaagacatctcggcgttctg (SEQ ID NO: 102)

TABLE 6 PCR Primers for Substitution Mutant AmaEcR Ligand Binding Domain ConstructionPRIMER MUTANT (SEQ ID NO:) PRIMER NUCLEOTIDE SEQUENCE (5′ TO 3′) G91A FPgtgatgatgctgagagctgcccggaaatatgatg (SEQ ID NO: 103) G91A RCPcatcatatttccgggcagctctcagcatcatcac (SEQ ID NO: 104) A105P FPacagattctatagtgtttcccaataaccagccgtacac (SEQ ID NO: 105) A105P RCPgtgtacggctggttattgggaaacactatagaatctgt (SEQ ID NO: 106)

The resulting PCR nucleic acid products encoding the mutant EcR ligandbinding domains were then each fused to a GAL4 DNA binding domain asdescribed in Examples 1.2, 1.4, 1.9 and 1.13 above. The GAL4/mutant EcRreceptor constructs were tested for activity by transfecting them intoNIH3T3 cells along with VP16/LmUSP-EF and pFRLuc in the presence ofsteroid or non-steroid ligand.

The resulting nucleic acids encoding the mutant EcR ligand bindingdomains were also each fused to a VP16 transactivation domain asdescribed in Examples 1.6 and 1.11 above. The VP16/mutant CfEcR-DEF andVP16/mutant DmEcR-CDEF receptor constructs were tested for activity bytransfecting them into L57 insect cells along with a6XEcRE/β-galactosidase reporter gene in the presence of20-hydroxyecdysone (20E).

Ligands:

The steroidal ligands muristeroneA, ponasterone A, α-ecdysone, and20-hydroxyecdysone were purchased from Sigma Chemical Company andInvitrogen. The non-steroidal ligands:N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine(GS™-E non-steroidal ligand);N′-tert-butyl-N′-(3,5-dimethylbenzoyl)-3-methoxy-2-methylbenzohydrazide(RH-2485); N-tert-butyl-N′-(4-ethylbenzoyl)-3,5-dimethylbenzohydrazide(RH-5992), andN′-tert-butyl-N′-(3,5-dimethylbenzoyl)-3,4-(1,2-ethylenedioxy)-2-methylbenzohydrazide(RH-125020) are synthetic stable ecdysteroid ligands synthesized at Rohmand Haas Company. All ligands were dissolved in DMSO and the finalconcentration of DMSO was maintained at 0.1% in both controls andtreatments. ³H-PonA and ³H-α-ecdysone were purchased from New EnglandNuclear. ³H-RH2485 was synthesized at Rohm and Haas Company.

Transfections:

DNAs corresponding to the various switch constructs outlined in Example1, specifically switches 1.1-1.13, were transfected into mouse NIH3T3cells (ATCC) or L57 cells (Dr. Peter Cherbas; Indiana University) asfollows. Standard methods for culture and maintenance of the cells werefollowed. Cells were harvested when they reached 50% confluency andplated in 6-, 12- or 24-well plates at 125,000, 50,000, or 25,000 cells,respectively, in 2.5, 1.0, or 0.5 ml of growth medium containing 10%fetal bovine serum (FBS), respectively. The next day, the cells wererinsed with growth medium and transfected for four hours. Superfect™(Qiagen Inc.) was used for 3T3 cells and Lipofectamine™(LifeTechnologies) was used for L57 cells as the transfection reagents.For 12-well plates, 4 μl of Superfect™ or Lipofectamine™ was mixed with100 μl of growth medium. One μg of reporter construct and 0.25 μg ofeach receptor construct of the receptor pair to be analyzed were addedto the transfection mix. A second reporter construct was added [pTKRL(Promega), 0.1 μg/transfection mix] that comprises a Renilla luciferasegene operably linked and placed under the control of a thymidine kinase(TK) constitutive promoter and was used for normalization. The contentsof the transfection mix were mixed in a vortex mixer and let stand atroom temperature for 30 minutes. At the end of incubation, thetransfection mix was added to the cells maintained in 400 μl growthmedium. The cells were maintained at 37° C. and 5% CO₂ for four hours.At the end of incubation, 500 μl of growth medium containing 20% FBS andeither dimethylsulfoxide (DMSO; control) or a DMSO solution of steroidalor non-steroidal ligand was added and the cells were maintained at 37°C. and 5% CO₂ for 48 hours. The cells were harvested and reporteractivity was assayed. The same procedure was followed for 6 and 24 wellplates as well except all the reagents were doubled for 6 well platesand reduced to half for 24-well plates.

Reporter Assays:

Cells were harvested 40 hours after adding ligands. 125 μl of passivelysis buffer (part of Dual-Luciferase™ reporter assay system fromPromega Corporation) were added to each well of the 24-well plate. Theplates were placed on a rotary shaker for 15 minutes. Twenty μl oflysate were assayed. Luciferase activity was measured usingDual-Luciferase™ reporter assay system from Promega Corporationfollowing the manufacturer's instructions. β-Galactosidase was measuredusing Galacto-Star™ assay kit from TROPIX following the manufacturer'sinstructions. All luciferase and β-galactosidase activities werenormalized using Renilla luciferase as a standard. Fold activities werecalculated by dividing normalized relative light units (“RLU”) in ligandtreated cells with normalized RLU in DMSO treated cells (untreatedcontrol).

Example 2

This Example describes the identification of improved non-steroidresponsive CfEcR ligand binding domain substitution mutants that exhibitincreased activity in response to non-steroidal ligand and decreasedactivity in response to steroidal ligand. Briefly, Applicants mutatedamino acid residues predicted to be critical for ecdysteroid bindinginto alanine and created GAL4/mutantCfEcR-DEF cDNA gene expressioncassettes as described in Example 1 above using the QuikchangePCR-mediated site-directed mutagenesis kit (Stratagene, La Jolla,Calif.). The mutated and the WT cDNAs were tested in GAL4-drivenluciferase reporter assays.

Transfections:

DNAs corresponding to the various switch constructs outlined in Example1, specifically switches 1.1-1.2, were transfected into mouse NIH3T3cells (ATCC) as follows. Cells were harvested when they reached 50%confluency and plated in 24 well plates at 12,500 cells/well in 0.5 mlof growth medium containing 10% fetal bovine serum (FBS). The next day,the cells were rinsed with growth medium and transfected for four hours.Superfect™ (Qiagen Inc.) was found to be the best transfection reagentfor 3T3 cells. Two μl of Superfect™ was mixed with 100 μl of growthmedium and 50 ng of either GAL4/wild-type EcR or Gal4/mutant EcRcassette, 50 ng of VP16/LmUSP-EF and 200 ng of pFRLuc were added to thetransfection mix. A second reporter construct was added [pTKRL(Promega), 0.05 μg/transfection mix] that comprises a Renilla luciferasegene operably linked and placed under the control of a thymidine kinase(TK) constitutive promoter and was used for normalization. The contentsof the transfection mix were mixed in a vortex mixer and let stand atroom temperature for 30 min. At the end of incubation, the transfectionmix was added to the cells maintained in 200 μl growth medium. The cellswere maintained at 37° C. and 5% CO₂ for four hours. At the end ofincubation, 250 μl of growth medium containing 20% FBS and eitherdimethylsulfoxide (DMSO; control) or a DMSO solution of 10 nM or 2.5 μMPonA steroidal ligand or GS™-E[N-(2-ethyl-3-methoxybenzoyl)N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine]non-steroidal ligand was added and the cells were maintained at 37° C.and 5% CO₂ for 40 hours. The cells were harvested and reporter activitywas assayed as described above. Fold activities were calculated bydividing normalized relative light units (“RLU”) in ligand treated cellswith normalized RLU in DMSO treated cells (untreated control).

Two amino acid residues were identified that, when substituted, yield amutant ecdysone receptor that exhibits increased activity in response toa non-steroid ligand and decreased activity in response to a steroidligand. The effect of alanine substitution at amino acid residue 52 or55 of SEQ ID NO: 1 on the activity of the mutated CfEcR-DEF receptor ispresented in Table 7 as a fold increase over Gal4/wild-type CfEcR-DEF(WT) switch activity.

TABLE 7 CfEcR-DEF Mutants that show increased non-steroid activity anddecreased steroid activity Fold increase over WT MUTANTS 2.5 μM GS ™-E2.5 μM PonA T52A 1.5 0.5 T55A 1.7 0.13

Example 3

This Example describes the identification of steroid responsive CfEcRligand binding domain substitution mutants that exhibit increasedactivity in response to steroidal ligand and significantly decreasedactivity in response to non-steroidal ligand. In an effort to identifysubstitution mutations in the CfEcR that increase steroidal ligandactivity, but decrease non-steroidal ligand activity, Applicants mutatedamino acid residues predicted to be critical for ecdysteroid binding andcreated GAL4/mutantCfEcR-DEF cDNA gene expression cassettes as describedin Example 1 above using PCR-mediated site-directed mutagenesis kit. Themutated and the WT cDNAs corresponding to the various switch constructsoutlined above in Examples 1.1 and 1.2 were made and tested inGAL4-driven luciferase reporter assays as described in Example 2 above.Fold activity was calculated by dividing RLUs in the presence of ligandwith RLUs in the absence of the ligand.

Specific amino acid residues were identified that, when substituted,yield a mutant ecdysone receptor that exhibits increased activity inresponse to a steroid ligand and decreased activity in response to anon-steroid ligand. The effect of an amino acid substitution at aminoacid residue 52 or 96 of SEQ ID NO: 1 and amino acid substitution atamino acid residues 96, 107 and 175 of SEQ ID NO: 1 on the activity ofthe mutated CfEcR-DEF receptor is presented in Table 8 as a foldincrease over Gal4/wild-type CfEcR-DEF (WT) switch activity.

TABLE 8 Mutants that show increased steroid and decreased non-steroidactivity Fold increase over WT 2.5 μM 2.5 μM 10 nM 10 nM MUTANTS GS ™EPonA GS ™E PonA T52L 0.26 3.4 V96A 0.35 408 V96T 0.018 45V96T/V107I/R175E 0.4 485.7

Example 4

This Example describes the identification of improved steroid andnon-steroid responsive CfEcR ligand binding domain substitution mutantsthat exhibit increased activity in response to both a steroidal ligandand a non-steroidal ligand. In an effort to identify substitutionmutations in the CfEcR that increase both steroidal and non-steroidalligand activity, Applicants mutated amino acid residues predicted to becritical for steroid binding and created GAL4/mutantCfEcR-DEF cDNA geneexpression cassettes as described in Example 1 above using PCR-mediatedsite-directed mutagenesis. The mutated and the WT cDNAs corresponding tothe various switch constructs outlined above in Examples 1.1 and 1.2were made and tested in GAL4-driven luciferase reporter assays asdescribed in Example 2 above. Fold activity was calculated by dividingRLUs in the presence of ligand with RLUs in the absence of the ligand.

Specific amino acid residues were identified that, when substituted,yield a mutant ecdysone receptor that exhibits increased activity inresponse to both non-steroid and steroid ligands. The effect of an aminoacid substitution at amino acid residue 52, 96, 107 or 175 of SEQ ID NO:1, amino acid substitution at amino acid residues 107 and 175 of SEQ IDNO: 1, amino acid substitution at amino acid residues 127 and 175 of SEQID NO: 1, amino acid substitution at amino acid residues 107 and 127 ofSEQ ID NO: 1, amino acid substitution at amino acid residues 107, 127and 175 of SEQ ID NO: 1, amino acid substitution at amino acid residues52, 107 and 175 of SEQ ID NO: 1 or amino acid substitution at amino acidresidues 96, 107 and 175 of SEQ ID NO: 1 on the activity of the mutatedCfEcR-DEF receptor is presented in Table 9 as a fold increase overGal4/wild-type CfEcR-DEF (WT) switch activity.

TABLE 9 Mutants that show increased steroid and non-steroid activityFold increase over WT 2.5 μM 2.5 μM 10 nM 10 nM MUTANTS GS ™E PonA GS ™EPonA T52V 17.3 35.7 T52I 8 8 V96D 3.07 3.1 V96M 122 3.37 V1071 12.4 26.6R175E 22.0 11.3 V107I/R175E 386.4 1194.4 Y127E/R175E 622.8 42.2V107I/Y127E 314.6 35.8 V107I/Y127E/R175E 124.3 122.3 T52V/V107I/R175E62.8 136.6 V96A/V107I/R175E 21.1 1005.1 T52A/V107I/R175E 2.3 20.3

Example 5

This Example describes the identification of non-steroid responsiveCfEcR ligand binding domain substitution mutants that exhibitsignificantly decreased activity in response to steroidal ligand but donot affect activity in response to non-steroidal ligand. In an effort toidentify substitution mutations in the CfEcR that decrease steroidalligand activity, but do not affect non-steroidal ligand activity,Applicants mutated amino acid residues predicted to be critical forecdysteroid binding and created GAL4/mutantCfEcR-DEF cDNA geneexpression cassettes as described in Example 1 above using PCR-mediatedsite-directed mutagenesis. The mutated and the WT cDNAs corresponding tothe various switch constructs outlined above in Examples 1.1 and 1.2were made and tested in GAL4-driven luciferase reporter assays asdescribed in Example 2 above. Fold activity was calculated by dividingRLUs in the presence of ligand with RLUs in the absence of the ligand.

Four amino acid residues were identified that, when substituted, yieldmutant ecdysone receptor that exhibit decreased activity in response toa steroid ligand and minimal effect on activity in response to anon-steroid ligand. The effect of an amino acid substitution at aminoacid residue 20, 58, 92, or 110 of SEQ ID NO: 1 on the activity of themutated CfEcR-DEF receptor is presented in Table 10 as a fold increaseover Gal4/wild-type CfEcR-DEF (WT) switch activity.

TABLE 10 Mutants that show decreased steroid activity, but non-steroidactivity is unaffected Fold increase over WT MUTANTS 2.5 μM GS ™E 2.5 μMPonA E20A 0.9 0.35 T58A 0.8 0.008 M92A 0.7 0.39 A110P 0.8 0.005

As described in Table 10, Applicants have identified point mutations inthe ligand binding domain of CfEcR that significantly reduce steroidbinding activity. CfEcR point mutants T58A and A110P essentiallyeliminated steroid binding activity. Interestingly, the non-steroidactivity of these point mutants was not significantly affected.

Example 6

Applicants have further characterized the non-steroid A110P CfEcRreceptor identified in Example 5 above. This Example demonstrates thatthe mutation of a critical alanine residue (A110) leads to thedisruption of steroid binding and hence transactivation by the EcR inthe presence of steroids. However, the binding as well astransactivation by non-steroids is not impaired.

Ligand Binding Assay

Applicants tested the A110P mutant CfEcR receptor in a steroid andnon-steroid ligand binding assay to confirm that steroid binding waseliminated. Briefly, PonA binding activity was determined using an invitro ligand binding assay (LBA). Steroid ligand binding assay (LBA) wasperformed using ³H-PonA (200 Ci/mmol). In vitro translatedGal4/wild-type or A110P mutant CfEcR-DEF and bacterial expressedGST-CfUSP-A/BCDEF were used in the assay. The assay was performed with 8μL of Gal4/wild-type or A110P mutant CfEcR-DEF, 2.5 uL ofGST-CfUSP-A/BCDEF, 1 μL of ³H-PonA, and 2 μL of unlabeled (“cold”) PonAas competitor in the presence of T buffer [90 mM Tris pH 8.0, 10 μM DTTcomprising Complete™ protease inhibitor cocktail used according to themanufacturer's instructions (Boehringer Mannheim)]. The reaction wascarried out at room temperature for 1 hour followed by the addition ofdextran-coated charcoal (Sigma). The mixture was centrifuged at 7000×gfor 10 minutes the amount of ³H-PonA in the supernatant was measured.The reactions were done in triplicate. The full-length WT EcR or itsA110P mutant were also in vitro translated and transcribed using the TNTsystem (Promega) according to the manufacturer's instructions and testedin in vitro ligand binding assays using 3H-RH2485, with cold 20E ornon-steroids (RH2485 and GS™-E) as competitors. In addition, 5 μL of thein vitro translations were assayed for translation efficiency usingSDS-PAGE following standard methods (Maniatis, 1989). The ligand bindingresults for both the wild-type and A110P mutant CfEcR-DEF receptors werecalculated and are shown in FIG. 1.

The two non-steroid ligands, RH2485 and GS™-E, and the steroid ligand20E tested were able to effectively compete with bound ³H-RH2485suggesting that they are able to bind the WT full-length EcR efficiently(see FIG. 1). However, when the binding of the same ligands by the A110Pmutant was examined, binding of the steroid 20E was completely disruptedbut the binding of the non-steroids was unaffected (see FIG. 1). Theseresults indicate that the lack of steroid binding in the case of theGAL4CfEcR fusion protein is not an artifact of the truncation or fusionand demonstrates that the A110P mutant CfEcR is a selective non-steroidreceptor.

Ligand Affinities of Mutant A110P CfEcR to Various Steroid andNon-Steroid Ligands:

The ligand binding affinities of the A110P GAL4/CfEcR mutant weremeasured by binding ³H-RH2485 and competing it with differentconcentrations of cold steroids or non-steroids. Briefly, 3H-RH2485 wasbound to in vitro translated full-length CfEcR and bacterially expressedCfUSP and competed with increasing concentrations of cold steroidal ornon-steroidal ligands. The reaction was carried out at room temperaturefor 1 hour followed by addition of activated dextran coated charcoal andcentrifugation at 7000×g for 10 minutes at 4° C. The residual ³H-RH2485in the supernatant after centrifugation was measured using ascintillation counter. The fraction bound (f bound) values weredetermined and plotted against the concentration of ligand (in mM). The1050 values were determined for each of the steroid PonA and MurA andnon-steroidN-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine(GS™-E non-steroidal ligand);N′-tert-butyl-N′-(3,5-dimethylbenzoyl)-3-methoxy-2-methylbenzohydrazide(RH-2485); N-tert-butyl-N′-(4-ethylbenzoyl)-3,5-dimethylbenzohydrazide(RH-5992), andN′-tert-butyl-N′-(3,5-dimethylbenzoyl)-3,4-(1,2-ethylenedioxy)-2-methylbenzohydrazide(RH-125020) ligands for the WT and the mutant was determined by plottingthe fraction bound against the concentration of each ligand.

As shown in Table 11, the 1050 values for steroids PonA and MurA wereincreased (more than 1 mM) in the case of the A110P mutant compared tothe observed nanomolar values for the WT receptor, suggesting that thebinding of steroids was impaired in the A110P mutant CfEcR. On the otherhand, non-steroid 1050 values were similar for both the A110P mutant andWT receptors (see Table 11). These results confirm Applicants' findingspresented in Example 5 above that the A110P substitution mutationresults in a non-steroid ecdysone receptor ligand binding domain thathas lost the ability to bind steroid ligand.

TABLE 11 IC50 values determined for wild-type and A110P mutantCfEcR-A/BCDEF using several steroidal and non-steroidal ligands.Wild-type IC50 (nM) A110P mutant IC50 (nM) Steroids: Ponasterone A345.95 >1 mM Muristerone A 423.99 >1 mM Non-Steroids: GS ™-E 85.26 12.88RH-5992 132.81 322.34 RH-2485 1.80 × 10³ 350.42 RH-125020 10.11 25.71

A110P in Truncated CfEcR or Full Length CfEcR Background

To eliminate the possibility that the loss of steroid activity may bedue to the truncated CfEcR-DEF receptor or an artifact of the GAL4fusion protein, Applicants introduced the A110P mutation into the fulllength (FL) CfEcR (CfEcR-A/BCDEF), fused it to the GAL4 DNA bindingdomain as described in Example 1.4, and comparatively assayed it inNIH3T3 cells in 24-well plates as described in Example 2 above with fulllength wild type CfEcR (Example 1.3) in combination with VP16/LmUSP-EFand pFREcRE Luc that comprised the luciferase reporter gene operativelylinked to six copies of the ecdysone response element (6× EcRE) and asynthetic TATAA. The transfected cells were grown in the presence of0.25 or 10 μM PonA or GS™-E and the reporter activity was measured at 40hours after adding ligand. The cells were harvested and the extractswere assayed for luciferase activity. The results are presented in FIG.2. The numbers on top of the bars indicate fold increase over DMSOlevels.

As shown in FIG. 2, the A110P mutation had a similar effect whenintroduced into the full-length ecdysone receptor in the context of anEcRE-driven reporter gene. Specifically, PonA activity was completelylost for the full length A110P CfEcR mutant. However, there was nosignificant difference in non-steroid activity between the full lengthWT CfEcR and the full-length A110P mutant CfEcR in the presence ofGST™-E. These results indicate that the non-responsiveness of the A110Pmutant to steroids observed with the GAL4-fusion CfEcR in Example 5 wasnot an artifact of the GAL4-fusion or truncation of EcR. Thus,Applicants have determined that the A110 amino acid residue is criticalfor steroidal activity in the full-length ecdysone receptor.

A110 Residue is Critical for Steroidal Activity in Insect Cells

In mammalian cells, the natural ligand of EcR, 20-hydroxyecdysone (20E)does not induce transactivation through a CfEcR-based gene expressionsystem. To determine whether the A110P mutant can respond to 20E,Applicants tested this A110P mutant-based gene expression system in anEcRE-driven reporter assay in insect L57 cells (a Drosophilamelanogaster cell line that lacks EcR isoform B, the Drosophilamelanogaster EcR isoform homolog of CfEcR isoform B from which the A110Pmutant is derived, however L57 cells still contain EcR isoform A). Themutation was introduced into the VP16/CfEcR-CDEF fusion protein andoperably linked to an baculovirus 1E1 promoter and L57 cells weretransfected with IE1VP16CfEcRCDEF (Example 1.5) or its A110P mutantversion DNA (Example 1.6) along with a pMK43.2 (3-galactosidase reportergene under the control of 6× ecdysone response elements (“6XEcRE”; thepMK43.2 construct was obtained from Michael Koelle at StanfordUniversity) in 24-well plates. Reporter activity for A110P mutant-basedgene expression system transactivation was measured after 40 hours oftreatment of the transfected cells with 0, 1, 10, 100, or 1000 nM 20E orGST™-E. The cells were harvested and the extracts were assayed forβ-galactosidase (β-gal) and luciferase activity. (3-Galactosidase wasmeasured using Galacto-Star™ assay kit from TROPIX following themanufacturer's instructions. The numbers on top of the bars indicatefold increase over DMSO levels. The results are presented in FIG. 3.

In insect cells, the wild-type CfEcR-based gene expression systeminduced β-gal activity in a dose dependent manner in response to both20E and GS™-E, whereas the A110P mutant-based gene expression systemtransactivated reporter gene expression in the presence of GS™-E,however, the L57 cells transfected with the A110P mutant showed slightlyincreased reporter gene activity in the presence of 20E (see FIG. 3).This low level activity in the presence of 20E is most likely due toendogenous activity of the EcR isoform A within the L57 cells sinceApplicants have demonstrated that the A110P mutant derived from CfEcRisoform B does not 20E (data not shown).

The A110P mutation had a similar effect when introduced into thefull-length receptor in the context of an EcRE-driven reporter gene inthe L57 cells indicating that the mutation has an analogous effect ininsect cells, presumably in the presence of insect transcriptionalco-factors (data not shown). These data confirm Applicants' results frommammalian cells and establish that the A110P mutation results in adrastic effect on the steroidal responsiveness of CfEcR but does notaffect the non-steroid responsiveness of CfEcR.

Example 7

Applicants' results presented above in Examples 5 and 6 describe theidentification of an alanine residue at position 110 that is a criticalresidue for steroid but not for non-steroid activity of the CfEcR ligandbinding domain. To further characterize the role of residue A110 inCfEcR steroid and non-steroid transactivation of reporter genes, a minilibrary of CfEcR-DEF receptors was prepared by mutating A110 usingdegenerate primers. These degenerate PCR primers (A110P random primerpairs comprising either SEQ ID NO: 83 and SEQ ID NO: 84 or SEQ ID NO: 85and SEQ ID NO: 86; see Table 4) were designed to replace A110 withvarious amino acid residues. The PCR mutagenesis conditions used were asdescribed above in Example 1. The resulting clones were sequenced toidentify the mutants.

Reporter Gene Transactivation of A110 Mutants

Four mutations: A110S, A110P, A110L, and A110M were obtained. These fourmutant and wild-type receptors were assayed in NIH3T3 cells. GAL4fusions of each of the four mutants or wild-type CfEcR-DEF receptor,VP16LmUSP-EF and pFRLUC were transfected into NIH3T3 cells, the cellswere grown in the presence of 0, 0.04, 0.2, 1, 5, or 25 μM PonA, MurA,N-(2-ethyl-3-methoxybenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butylhydrazine(GS™-E non-steroidal ligand), orN′-tert-butyl-N′-(3,5-dimethylbenzoyl)-3,4-(1,2-ethylenedioxy)-2-methylbenzohydrazide(RH-125020) for 48 hours and the reporter activity was measured. Asshown in FIG. 4, the wild-type ecdysone receptor showed reporteractivity in the presence of both steroids and non-steroids. However, allmutant receptors showed reporter activity only in the presence ofnon-steroid ligands but not in the presence of steroid ligands. TheA110P mutant exhibited similar non-steroid activity compared towild-type receptor, however the A110S, A110L and A110M mutantsdemonstrated lower sensitivity and no detectable transactivation at thetwo lowest concentrations. These results confirm that an A110substitution mutant EcR ligand binding domain is characterized by asignificantly reduced response to steroids but remains responsive tonon-steroids.

Ligand Binding by the A110 Mutants:

Applicants have performed ligand binding assays using ³H-PonA or³H-RH2485(N′-tert-butyl-N′-(3,5-dimethylbenzoyl)-3-methoxy-2-methylbenzohydrazide)radioactively-labeled ligands to determine if the differences in ligandresponse of substitution mutant and wild-type ecdysone receptors are dueto differences at the level of ligand binding. The CfEcR wild-type andmutant receptors were in vitro transcribed and translated and assayed inthe presence of bacterially expressed GST-CfUSP-A/BCDEF (full length)PonA binding to A110P mutant was undetectable while other A110substitution mutants showed 5-10% of wild-type receptor binding (seeFIG. 5). Wild-type EcR and all mutants tested showed similar binding toRH-2485, a close analog of GS™-E (see FIG. 6). It may be that theinflexible proline residue of the A110P mutant hinders binding ofsteroidal but not non-steroidal ligands.

The in vitro translated proteins were analyzed by SDS-PAGE and werefound to be translated similar to the WT receptor, indicating that thedifferences in binding observed between the mutants and wild-typereceptor are not due to variation in the amount of proteins present inassay (data not shown). The ligand binding activity correlates with thereporter gene activity in most cases, providing further evidence ofApplicants' discovery that the A110 residue plays a critical role in thebinding of ecdysteroids, but not non-steroids.

All of the A110 substitution mutants were impaired in steroid binding aswell as in their ability to transactivate reporter genes in the presenceof steroids in mammalian and insect cells. These mutants maintainedwild-type levels of non-steroid binding and reporter genetransactivation in the presence of non-steroids.

The A110 residue, found adjoining the predicted β-sheet between helix 5and 6, is highly conserved in EcRs from various species of insects, andin RXR, progesterone receptor (PR) and estrogen receptor (ER) furtherunderlining that this residue is critical for ligand binding and thustransactivation. In addition, residue A110 is flanked by other highlyconserved residues, some of which may be also critical for ligandbinding and/or transactivation. Close comparison of thethree-dimensional structures of nuclear receptors show that there aremajor structural changes even in the conserved LBD among differentnuclear receptors. Important changes are observed not only betweendifferent nuclear receptor structures, but also complexes of the samereceptor when bound to natural and synthetic ligands as in the case ofER. The binding of steroid and non-steroids may reflect a similarsituation. The homology models generated suggest that the binding of thetwo ligands is different, in terms of the helices involved and contactresidues. Close examination of the transactivation assay resultssuggests that the dose responses are slightly different in the twosituations. The steroids are less active at lower concentrations whilethe non-steroids induced activity is several fold higher. However, athigher doses the steroidal and non-steroidal activities are similar. Thehigher activity of non-steroids at lower concentrations may reflecthigher affinity of the non-steroids to the EcR. It has been suggestedthat the presence of the tert-butyl group allows some non-steroids toform extensive van der Waals contacts with the EcR LBD and thus fits ina groove that is not occupied by ecdysteroids. This may explain to someextent the differences seen in the activities of the steroids and thenon-steroids in reporter gene assays at low ligand concentrations. Themechanism by which binding affects conformational changes and thustransactivation potential is yet unknown. However, in the case of theER, the binding of agonists and antagonists have been shown to inducedifferent conformational changes resulting in displacement of the helix12. Helix 12 plays an active role in the recruitment and interaction ofcoactivators to the receptor. In the case of agonists likediethylstilbestrol (DES), coactivator GRIP1 binds to a hydrophobicgroove on the surface of the LBD formed by helices 3, 4, 5, and 12 andthe turn between helices 3 and 4. On the other hand, in the presence apartial antagonist, 4-hydroxytamoxifen (OHT), helix 12 blocks thecoactivator recognition groove by mimicking the interactions of GRIP NRbox with the LBD. The binding of steroid and the non-steroids could alsoinduce subtle conformational changes which affects coactivatorrecruitment and thus transactivation.

The A110 residue appears to interact with the side chain of the steroidligand. The introduction of bulkier or inflexible residues in thisposition would potentially disrupt these interactions and thus dockingof the ligand to the LBD. This in turn results in non-activation of theEcR. The identification of a mutant that results in disruption ofecdysteroid binding without affecting non-ecdysteroid binding andactivation provides a means for systematic evolution of the EcR todevelop an ecdysone inducible system that can be precisely regulated foruse in mammalian systems.

Example 8

This Example describes the identification of CfEcR ligand binding domainsubstitution mutants that exhibit decreased activity in response to botha steroidal ligand and a non-steroidal ligand. These substitutionmutants are useful in ligand screening assays for orthogonal ligandidentification. In an effort to identify substitution mutations in theCfEcR that decrease both steroidal and non-steroidal ligand activity,Applicants mutated amino acid residues predicted to be critical forecdysteroid binding and created GAL4/mutantCfEcR-DEF cDNA geneexpression cassettes as described in Example 1 above using PCR-mediatedsite-directed mutagenesis. The mutated and the WT cDNAs corresponding tothe various switch constructs outlined above in Examples 1.1 and 1.2were made and tested in GAL4-driven luciferase reporter assays asdescribed in Example 2 above. Fold activity was calculated by dividingRLUs in the presence of ligand with RLUs in the absence of the ligand.

Seventeen amino acid residues were identified that, when substituted,yield a mutant ecdysone receptor that exhibits decreased activity inresponse to both non-steroid and steroid ligands. The effect of an aminoacid substitution at amino acid residue 21, 48, 51, 59, 62, 93, 95, 109,120, 123, 125, 218, 219, 223, 230, 234, or 238 of SEQ ID NO: 1 on theactivity of the mutated CfEcR-DEF receptor is presented in Table 12 as afold increase over Gal4/wild-type CfEcR-DEF (WT) switch activity. Inaddition, two double mutants (R95A/A110P and M218A/C219A) and one triplemutant (V107I/A110P/R175E) were made and were also identified as mutatedCfEcR-DEF receptors that exhibit decreased activity in response to bothnon-steroid and steroid ligands (see Table 12).

TABLE 12 Mutants that show decreased steroid and non-steroid activityFold increase over WT 2.5 μM 2.5 μM 10 nM 10 nM MUTANTS GS ™-E PonAGS ™-E PonA Q21A 0.32 0.37 F48A 0.007 0.007 I51A 0.003 0.004 V59A 0.470.002 I62A 0.12 0.004 M93A 0.46 0.07 R95A 0.4 0.006 F109A 0.22 0.005Y120A 0.001 0.006 A123F 0.09 0.005 M125A 0.005 0.007 M218A 0.001 0.001C219A 0.001 0.001 L223A 0.118 0.007 L230A 0.001 0.006 L234A 0.001 0.006W238A 0.002 0.013 R95A/A110P 0.4 0.007 M218A/C219A 0.001 0.001 0.345 nd*V107I/A110P/R175E *Not detectable

Example 9

This Example describes the introduction of substitution mutations withinthe Drosophila melanogaster EcR (DmEcR) at amino acid residues withinthe DmEcR ligand binding domain that are analogous to the CfEcR ligandbinding domain substitution mutants identified above. Specifically,substitution mutations were introduced at DmEcR amino acid residues 107,121, 213, and 217 of SEQ ID NO: 2, corresponding to CfEcR amino acidresidues 110, 124, 211, and 219 of SEQ ID NO: 1, respectively.

Applicants mutated amino acid residues predicted to be critical forecdysteroid binding and created GAL4/mutantDmEcR-CDEF cDNA geneexpression cassettes as described in Example 1 above using PCR-mediatedsite-directed mutagenesis. The mutated and the WT cDNAs corresponding tothe various switch constructs outlined above in Examples 1.8 and 1.9were made and tested in reporter assays in NIH3T3 cells as described inExample 2. Each GAL4/DmEcR-CDEF construct, VP16/LmUSP-EF, and pFRLUCwere transfected into NIH3T3 cells and the transfected cells weretreated with 2.5 μM GS™-E or Ponasterone A. The cells were harvested andthe reporter activity was measured at 48 hours after addition of ligand.The fold induction was calculated by dividing reporter activity in thepresence of ligand with the reporter activity in the absence of ligand.From the fold induction, percent wild-type activity was calculated foreach mutant. The results are presented in Table 13.

TABLE 13 GAL4/DmEcR-CDEF wild-type and Substitution Mutants G121R,G121L, G217A, and C217 S tested for transactivation in NIH3T3 cells.Fold increase over WT: DmEcR-CDEF Mutant 2.5 μM Ponasterone A 2.5 μMGS ™-E G121R 0.05 0.0075 G121L 0.001 0.008 C217A 0.022 0.008 C217S0.0064 0.014

As seen in Table 13, both non-steroid and steroid activities weredecreased significantly when the DmEcR ligand binding domain was mutatedat amino acid residues 121 or 217, indicating that these residues areimportant residues in the ligand binding pocket of DmEcR.

The wild-type and mutant DmEcR-CDEF receptors were also used to make VP16/wild-type or mutantDmEcR-CDEF constructs as described in Example 1.10and 1.11. VP16DmEcR-CDEF and a 6XEcREβ-gal reporter were transfectedinto L57 cells and the transfected cells were treated with 1 uM20-hydroxyecdysone (20E) or GST™-E. The cells were harvested, lysed andthe reporter activity was measured as described above in Example 6. Thefold induction was calculated by dividing reporter activity in thepresence of ligand with the reporter activity in the absence of ligand.From the fold induction, percent wild-type activity was calculated foreach mutant. The results are presented in Table 14.

TABLE 14 VP16/DmEcR-CDEF wild-type and Substitution Mutants A107P,G121R, G121L, N213A, G217A, and C217 S tested for transactivation ininsect L57 cells. Fold increase over WT: DmEcR-CDEF Mutant 1 μM20-hydroxyecdysone 1 μM GS ™-E A107P 0.09 0.9 G121R 0.5 0.92 G121L 0.090.15 N213A 0.01 0.08 C217A 0.48 0.70 C217S 0.39 0.92

The A107P mutation of DmEcR caused the loss of most steroid activity buthad very little effect on non-steroid activity. The G121R and C217Smutations of DmEcR resulted in 50% and 61% reductions respectively insteroid activity but minimal effect on non-steroid activity. The C217Amutation of DmEcR resulted in reduced non-steroid and steroidactivities, and the DmEcR mutants G121L and N213A lost sensitivity toboth steroids and non-steroids, indicating that these residues areinvolved in binding to both steroids and non-steroids.

Example 10

This Example describes the introduction of substitution mutations withinthe Amblyomma americanum EcR (AmaEcR) at amino acid residues within theAmaEcR ligand binding domain that are analogous to the CfEcR ligandbinding domain substitution mutants identified above. Specifically,substitution mutations were introduced at AmaEcR amino acid residues 91and 105 of SEQ ID NO: 3, corresponding to CfEcR amino acid residues 96and 110 of SEQ ID NO: 1, respectively.

Applicants mutated amino acid residues predicted to be critical forecdysteroid binding and created GAL4/mutantAmaEcR-DEF cDNA geneexpression cassettes as described in Example 1 above using PCR-mediatedsite-directed mutagenesis. The mutated and the WT cDNAs corresponding tothe various switch constructs outlined above in Examples 1.12 and 1.13were tested in GAL4-driven luciferase reporter assays in NIH3T3 cells asdescribed in Example 2. GAL4/AmaEcR-DEF, VP16LmUSP-EF and pFRLUC weretransfected into NIH3T3 cells and the transfected cells were treatedwith either 0.2 μM Ponasterone A steroid ligand or 1 μM GS™-Enon-steroid ligand. The cells were harvested and the reporter activitywas measured at 48 hours after addition of ligand. The fold inductionwas calculated by dividing reporter activity in the presence of ligandwith the reporter activity in the absence of ligand. From the foldinduction, percent wild-type activity was calculated for each mutant.The results are presented in Table 15.

TABLE 15 AmaEcR-DEF Substitution Mutants at G91 and A105 in NIH3T3cells. Fold increase over WT: AmaEcR-DEF Mutant 0.2 μM Ponasterone A 1μM GS ™-E G91A 1.29 1.22 A105P 0.11 0.01

The G91A mutation of AmaEcR at the homologous amino acid residueposition of V96 in CfEcR resulted in increased steroid and non-steroidactivities. The A105P mutation of AmaEcR at the homologous amino acidresidue position of A110 of CfEcR caused the loss of most steroidactivity and essentially eliminated non-steroid activity.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

1.-20. (canceled)
 21. A method of modulating the expression of a gene ina host cell, said method comprising: a) introducing into the host cell agene expression modulation system comprising a recombinant geneexpression cassette that is capable of being expressed in a host cell,said recombinant gene expression cassette comprising a polynucleotidethat encodes a polypeptide comprising: i) a transactivation domain; ii)a DNA-binding domain that recognizes a response element associated witha gene whose expression is to be modulated; and iii) a nuclear receptorligand binding domain comprising a substitution mutation-selected fromthe group consisting of: (1) a substitution mutation at one or more ofamino acid residues 20, 21, 48, 51, 52, 55, 58, 59, 61, 62, 92, 93, 95,96, 107, 109, 110, 120, 123, 125, 175, 218, 219, 223, 230, 234, or 238of SEQ ID NO: 1; (2) a substitution mutation at each of amino acidresidues 95 and 110 of SEQ ID NO: 1; (3) a substitution mutation at eachof amino acid residues 218 and 219 of SEQ ID NO: 1; (4) a substitutionmutation at each of amino acid residues 107 and 175 of SEQ ID NO: 1; (5)a substitution, mutation at each of amino acid residues 127 and 175 ofSEQ ID NO: 1; (6) a substitution mutation at each of amino acid residues107 and 127 of SEQ ID NO: 1; (7) a substitution mutation, at each ofamino acid residues 107, 127 and 175 of SEQ ID NO: 1; (8) a substitutionmutation at each of amino acid residues 52, 107 and 175 of SEQ ID NO: 1;(9) a substitution mutation at each of amino acid residues 96, 107 and175 of SEQ ID NO: 1; and (10) a substitution mutation at each of aminoacid residues 107, 110 and 175 of SEQ ID NO: 1, wherein said nuclearreceptor ligand and binding domain is capable of binding adiacylhydrazine; and b) introducing into the host cell a ligand; whereinthe gene to be modulated is a component of a gene expression cassettecomprising: i) a response element recognized by the DNA binding domain;ii) a promoter that is activated by the transactivation domain; and iii)a gene whose expression is to be modulated; whereby upon introduction ofthe ligand into the host cell, expression of the gene of b)iii) ismodulated.
 22. The method according to claim 21, wherein the ligand isa) a compound of the formula:

wherein: E is a (C₄-C₆)alkyl containing a tertiary carbon or acyano(C₃-C₅)alkyl containing a tertiary carbon; R¹ is H, Me, Et, i-Pr,F, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN,C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF₂CF₃,CH═CHCN, allyl, azido, SCN, or SCHF₂; R² is H, Me, Et, n-Pr, i-Pr,formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C≡CH, 1propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe₂,NEt₂, SMe, SEt, SOCF₃, OCF₂CF₂H, COEt, cyclopropyl, CF₂CF₃, CH═CHCN,allyl, azido, OCF₃, OCHF₂, O-i-Pr, SCN, SCHF₂, SOMe, NH—CN, or joinedwith R³ and the phenyl carbons to which R² and R³ are attached to forman ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to aphenyl carbon, or a dihydropyryl ring with the oxygen adjacent to aphenyl carbon; R³ is H, Et, or joined with R² and the phenyl carbons towhich R² and R³ are attached to form an ethylenedioxy, a dihydrofurylring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ringwith the oxygen adjacent to a phenyl carbon; R⁴, R⁵, and R⁶ areindependently H, Me, Et, F, Cl, Br, formyl, CF₃, CHF₂, CHCl₂, CH₂F,CH₂Cl, CH₂OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, orSEt; or b) an ecdysone, 20-hydroxyecdysone, ponasterone A, muristeroneA, an oxysterol, a 22(R) hydroxycholesterol, 24(S) hydroxycholesterol,25-epoxycholesterol, T0901317,5-alpha-6-alpha-epoxycholesterol-3-sulfate, 7-ketocholesterol-3-sulfate,farnesol, a bile acid, a 1,1-biphosphonate ester, or a Juvenile hormoneIII.
 23. The method of claim 21, further comprising introducing into thehost cell a second ligand, wherein the second ligand is 9-cis-retinoicacid or a synthetic analog of a retinoic acid.
 24. (canceled) 25.(canceled)
 26. The method of claim 21, wherein one or more of said aminoacid substitution mutations is selected from the group consisting ofE20A, Q21A, F48A, I51A, T52A, T52V, T52I, T52L, T55A, T58A, V59A, L61A,I62A, M92A, M93A, R95A, V96A, V96T, V96D, V96M, V107I, F109A, A110P,A110S, A110M, A110L, Y120A, A123F, M125A, R175E, M218A, C219A, L223A,L230A, L234A, W238A, R95A/A110P, M218A/C219A, V107I/R175E, Y127E/R175E,V107I/Y127E, V107I/Y127E/R175E, T52V/V107I/R175E, V96A/V107I/R175E,52A/V107I/R175E, V96T/V107I/R175E, and V107I/A110P/R175E.
 27. The methodof claim 21, wherein said substitution mutation is at amino acidresidues 107 and
 127. 28. The method of claim 27, wherein saidsubstitution mutation at amino acid residue 107 is V107I, and thesubstitution mutation at amino acid residue 127 is Y127E.
 29. The methodof claim 27, wherein said recombinant gene expression modulation systemexhibits increased activity in response to a non-steroidal ligand,relative to a gene expression modulation system that contains saidnuclear receptor ligand binding domain in which amino acid residues 107and 127 of SEQ ID NO: 1 are not mutated.
 30. The method of claim 27,wherein: said transactivation domain is a VP16 transactivation domainsaid DNA-binding domain is a GAL4 DNA-binding domain that recognizessaid response element; said nuclear receptor ligand binding domain is aChoristoneura fumiferana ecdysone receptor ligand binding domain; andwherein said polynucleotide further encodes a chimeric retinoic Xreceptor ligand binding domain comprising a first polypeptide fragmentand a second polypeptide fragment, wherein the first polypeptidefragment is a vertebrate retinoid X receptor ligand binding domainfragment, and the second polypeptide fragment is an invertebrateretinoid X receptor ligand binding domain fragment.
 31. The method ofclaim 21, wherein said DNA binding domain is selected from the groupconsisting of a GAL4 DNA binding domain, a LexA DNA binding domain, atranscription factor DNA binding domain, a steroid/thyroid hormonenuclear receptor superfamily member DNA binding domain and a bacterialLacZ DNA binding domain.
 32. The method of claim 21, wherein saidtransactivation domain is selected from the group consisting of asteroid/thyroid hormone nuclear receptor transactivation domain, apolyglutamine transactivation domain, a basic or acidic amino acidtransactivation domain, a VP16 transactivation domain, a GAL4transactivation domain, an NF-κB transactivation domain, a p65transactivation domain and a BP42 transactivation domain.
 33. The methodof claim 21, wherein said recombinant gene expression system iscontained within a vector.
 34. The method of claim 33, wherein saidvector is a plasmid.
 35. The method of claim 33, wherein said vector isan expression vector.
 36. The vector of claim 33, wherein said vector isa viral vector.
 37. The vector of claim 36, wherein said viral vector isan adenovirus vector.
 38. The method of claim 21, wherein said host cellis selected from the group consisting of a bacterial cell, a fungalcell, a yeast cell, a plant cell, an animal cell, a mammalian cell, amouse cell, and a human cell.
 39. The method of claim 21, wherein saidhost cell is selected from the group consisting of an Aspergillus cell,a Trichoderma cell, a Saccharomyces cell, a Pichia cell, a Candida cell,and a Hansenula cell.
 40. The method of claim 21, wherein said host cellis selected from the group consisting of a Synechocystis cell, aSynechococcus cell, a Salmonella cell, a Bacillus cell, an Acinetobactercell, a Rhodococcus cell, a Streptomyces cell, an Escherichia cell, aPseudomonas cell, a Methylomonas cell, a Methylobacter cell, anAlcaligenes cell, a Synechocystis cell, an Anabaena cell, a Thiobacilluscell, a Methanobacterium cell and a Klebsiella cell.
 41. The method ofclaim 21, wherein said host cell is a plant cell.
 42. The method ofclaim 41, wherein said plant cell is selected from the group consistingof an apple cell, an Arabidopsis cell, a bajra cell, a banana cell, abarley cell, a bean cell, a beet cell, a blackgram cell, a chickpeacell, a chili cell, a cucumber cell, an eggplant cell, a favabean cell,a maize cell, a melon cell, a millet cell, a mungbean cell, an oat cell,an okra cell, a Panicum cell, a papaya cell, a peanut cell, a pea cell,a pepper cell, a pigeonpea cell, a pineapple cell, a Phaseolus cell, apotato cell, a pumpkin cell, a rice cell, a sorghum cell, a soybeancell, a squash cell, a sugarcane cell, a sugarbeet cell, a sunflowercell, a sweet potato cell, a tea cell, a tomato cell, a tobacco cell, awatermelon cell, and a wheat cell.
 43. The method of claim 38, whereinsaid host cell is a mammalian cell.
 44. The method of claim 43, whereinsaid mammalian cell is selected from the group consisting of a hamstercell, a mouse cell, a rat cell, a rabbit cell, a cat cell, a dog cell, abovine cell, a goat cell, a cow cell, a pig cell, a horse cell, a sheepcell, a monkey cell, a chimpanzee cell, and a human cell.
 45. The methodof claim 44, wherein said mammalian cell is a human cell.
 46. The methodof claim 21, wherein said host cell is isolated.